Research ArticleGlutamine Supplementation Attenuates Expressions ofAdhesion Molecules and Chemokine Receptors on T Cells ina Murine Model of Acute Colitis

Yu-Chen Hou1 Jin-Ming Wu1 Ming-Yang Wang1 Ming-Hsun Wu1 Kuen-Yuan Chen1

Sung-Ling Yeh2 and Ming-Tsan Lin13

1 Department of Surgery National Taiwan University Hospital Taipei 100 Taiwan2 School of Nutrition and Health Sciences Taipei Medical University Taipei 110 Taiwan3Department of Primary Care Medicine College of Medicine National Taiwan University Taipei 100 Taiwan

Correspondence should be addressed to Ming-Tsan Lin linmtntuedutw

Received 7 January 2014 Revised 21 March 2014 Accepted 7 April 2014 Published 7 May 2014

Academic Editor H Barbaros Oral

Copyright copy 2014 Yu-Chen Hou et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Migration of T cells into the colon plays a major role in the pathogenesis in inflammatory bowel disease This studyinvestigated the effects of glutamine (Gln) supplementation on chemokine receptors and adhesion molecules expressed by T cellsin mice with dextran sulfate sodium- (DSS-) induced colitisMethods C57BL6 mice were fed either a standard diet or a Gln dietreplacing 25 of the total nitrogen After being fed the diets for 5 days half of the mice from both groups were given 15 DSS indrinking water to induce colitis Mice were killed after 5 days of DSS exposure Results DSS colitis resulted in higher expressionlevels of P-selectin glycoprotein ligand- (PSGL-) 1 leukocyte function-associated antigen- (LFA-) 1 and C-C chemokine receptortype 9 (CCR9) by T helper (Th) and cytotoxic T (Tc) cells and mRNA levels of endothelial adhesion molecules in colons wereupregulated Gln supplementation decreased expressions of PSGL-1 LFA-1 and CCR9 by Th cells Colonic gene expressions ofendothelial adhesion molecules were also lower in Gln-colitis mice Histological finding showed that colon infiltrating Th cellswere less in the DSS group with Gln administration Conclusions Gln supplementation may ameliorate the inflammation of colitispossibly via suppression of T cell migration

1 Introduction

Inflammatory bowel disease (IBD) which includes Crohnrsquosdisease (CD) and ulcerative colitis (UC) is a relapsing andremitting disorder characterized by chronic inflammation ofthe gastrointestinal (GI) tract The etiology of IBD remainsunclear however both CD and UC are associated withenhanced leukocyte trafficking to the inflamed intestine [12] Previous studies indicated that effector T cells includingCD4-positive T helper (Th) cells and CD8-positivecytotoxicT (Tc) cells play pivotal roles in the pathogenesis of mucosallesions and chronic intestinal inflammation [3ndash5] Blockageof lymphocyte migration to mucosal sites has thereforebecome a potential therapeutic strategy for IBD [6]

Lymphocytes migrate to specific tissues via a multistepprocess which is strictly regulated by adhesion moleculesand chemokine receptors Adhesion molecules present on

the vascular endothelium and lymphocytes participate inthe tethering rolling and adhesion of lymphocytes [7] P-selectin and E-selectin appearing on activated endothelialcells interact with P-selectin glycoprotein ligand- (PSGL-) 1expressed by lymphocytes which promotes the initial teth-ering and subsequent rolling of lymphocytes over vesselwalls Integrins participate in rolling and firm adhesion oflymphocytes Lymphocytes expressing 12057241205737 integrins roll onmucosal addressin cell adhesion molecule- (MAdCAM-) 1which is required for homing of lymphocytes to intestinalsites [8] Lymphocyte arrest mediated by integrins suchas lymphocyte function-associated antigen- (LFA-) 1 (alsoknown as CD11aCD18 and 1205721198711205732 integrins) which inter-acts with their endothelial-cell ligands intercellular adhesionmolecule- (ICAM-) 1 precedes extravasation into the under-lying tissue [9]

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 837107 14 pageshttpdxdoiorg1011552014837107

2 Mediators of Inflammation

Chemokines are small peptides which bind to chemokinereceptors expressed on leukocytes and function as chemoat-tractants They regulate lymphocyte homing to secondarylymphoid organs and transmigration into tissues by forminga chemokine concentration gradient which attracts lympho-cytes to move towards an increasing concentration [10]Dysregulation of chemokines and chemokine receptors wasimplicated in various autoimmune diseases including IBD[11 12] Recent studies indicated that C-C chemokine receptortype 9 (CCR9) and 12057241205737 integrins are required for thelocalization of lymphocytes to the GI mucosa [13] Agentsdeveloped for disrupting actions of CCR9 and 12057241205737 integrinsshowed promising results in IBD clinical trials [6]

Glutamine (Gln) is an immunomodulatory nutrientwhich is widely used in clinical practice [14] Previous studiesshowed that Gln treatment has beneficial effects in differentexperimental models of colitis Gln attenuates the expressionof proinflammatory mediators [15] and improves outcomeswhich may be due to upregulation of heat shock proteins(HSPs) [16] Recent work in our laboratory demonstratedthat pretreatment with Gln suppresses cytokine expression ofTh cells and ameliorates the severity of acute dextran sulfatesodium- (DSS-) induced colitis [17] However whether thebeneficial effects of Gln are mediated by modulating lym-phocyte trafficking in colitis is still unclear Therefore weinvestigated the influence of dietary Gln supplementation onT cell adhesion molecules and CCR9 expression in mice withDSS-induced acute colitis

2 Materials and Methods

21 Study Protocols Six-week-old male C57BL6 mice wereused in this study Conventional mice were maintained ina temperature- and humidity-controlled room and were feda standard chow diet ad libitum before the study Care oflaboratory animals was in full compliance with the Guide forthe Care and Use of Laboratory Animals (National ResearchCouncil 1996) and protocols were approved by the Institu-tional Animal Care and Use Committee of Taipei MedicalUniversity

22 Experimental Design After 1 week of acclimation 40mice were randomly assigned to a control group or a Glngroup in this study with 20 mice in each group Mice in thecontrol group were fed a common semipurified diet whilethe Gln group received a diet in which part of the caseinwas replaced with Gln Gln provided 25 of the total aminoacid nitrogen This amount of Gln was proven to have animmunomodulatory effect in rodents [18ndash20] Two diets wereformulated to be isonitrogenous and isoenergetic (Table 1)After 5 d of being fed the diets mice in the control and Glngroups were further divided into 2 respective subgroups Onesubgroup was given distilled water while the other subgroupreceived 15 (wtvol) DSS (MW40 kDa MP BiomedicalsSolon OH USA) in the drinking water for 5 d to inducecolitis A flow diagram of the study design is shown inFigure 1 There were 4 groups in this study control diet withdistilled water (C group) Gln diet with distilled water (Ggroup) control diet with DSS water (DC group) and Gln

Table 1 Composition of the semipurified diets

Component Control diet Gln dietgkg

Soybean oil 100 100Casein 200 150Glutamine 0 417Salt mixturea 35 35Vitamin mixtureb 10 10Methyl cellulose 31 31Choline bitartrate 25 25Methionine 3 3Corn starch 6268 6185aThe salt mixture contained the following (mgg) calcium phosphatediabasic 500 sodium chloride 74 potassium sulfate 52 potassium citratemonohydrate 20 magnesium oxide 24 manganese carbonate 35 ferriccitrate 6 zinc carbonate 16 curpric carbonate 03 potassium iodate 001sodium selenite 001 and chromium potassium sulfate 055bThe vitamin mixture contained the following (mgg) thiamin hydrochlo-ride 06 riboflavin 06 pyridoxine hydrochloride 07 nicotinic acid 3calcium pantothenate 16 D-biotin 005 cyanocobalamin 0001 retinylpalmitate 16 DL-120572-tocopherol acetate 20 cholecalciferol 025 andmenaquinone 0005

Sacrifice(days)

Induction of

Water

DCDG

Con dietColitis groups

C Con dietGln diet

Gln diet

G

Healthygroups Water

15 DSS15 DSS

D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 D10

colitis (n = 10per group)

control

Figure 1 Flow diagram of the study design

diet withDSSwater (DG group)The respective experimentaldiets were given during the DSS exposure period Bodyweights (BWs) were recorded daily and all mice had freeaccess to food and water throughout the study At the endof the experiment mice were anesthetized and sacrificed bycardiac puncture Fresh blood samples were collected inheparinized tubes for measurements of the leukocyte popu-lation Mesenteric lymph nodes (MLNs) were removed andprocessed for further analysis by flow cytometry The colonwas cut close to the ileocecal valve and its length and weightwere measured Sections (1 cm) of the distal colon were cutColon tissues were fixed with buffered 4 paraformaldehydefor an immunohistochemical analysis

23 Blood Leukocyte Distribution A five-color flow cyto-metric analysis was performed to determine the distributionof peripheral blood leukocytes Antibodies against mouseleukocyte surface antigens were added to 100 120583L aliquots ofwhole blood The antibodies used to detect different sub-sets of leukocytes were as follows PerCP-conjugated anti-CD45 (Biolegend San Diego CA USA) for leukocytes PE-conjugated anti-F480 (eBioscience SanDiego CA USA) for

Mediators of Inflammation 3

monocytesmacrophages FITC-conjugated anti-Ly6G (BDBiosciences San Jose CA USA) for neutrophils APC-con-jugated anti-CD3120576 (eBioscience) for T cells and Pacific blue-conjugated anti-CD19 (Biolegend) for B cells Antibodieswere used at the concentration recommended by manufac-turer After a 30min incubation at 4∘C in the dark red bloodcells were lysed and cells were suspended in staining bufferand then analyzed with a FACS Canto II flow cytometer (BDBiosciences) CD45-positive cells were gated and results arepresented as a percentage of specific CD-marker-expressingcells in blood leukocytes Representative flow cytometry plotsare shown in Figure 2(a)

24 Lymphocyte Populations inMLNs Cell suspensions fromMLNs were obtained by passing the tissues through anylon cell strainer with a 40 120583m pore size (BD Biosciences)in RPMI1640 medium (Biological Industries Kibbutz BeitHaemek Israel) After centrifugation at 300timesg for 10minpelletedMLN cells were suspended in 1mL of staining bufferOne hundred microliters of cell suspension was incubatedwith APC-conjugated anti-CD3120576 (eBioscience) and Pacificblue-conjugated anti-CD19 (Biolegend) for 30min at 4∘Cin the dark Stained cells were washed and resuspended instaining buffer to measure the lymphocyte population byflow cytometry Percentages of T and B lymphocytes weredetermined by CD3120576- and CD19-expressing cells in MLNcells Representative flow cytometry plots are shown inFigure 2(b)

25 Expressions of Adhesion Molecules and Chemokine Recep-tors by T Cells Whole blood and MLNs were used to ana-lyze adhesion molecule- and chemokine receptor-express-ing T cells Whole blood and MLN cells obtained asdescribed above were split into 2 vials with 100 120583L ineach aliquot and these were incubated with Pacific blue-conjugated anti-CD4 (BD Biosciences) or Pacific blue-conjugated anti-CD8 antibodies (Biolegend) To investigateexpressions of adhesion molecules and chemokine receptorsPE-conjugated anti-PSGL-1 antibodies (BD Biosciences)APC-conjugated anti-12057241205737 integrin FITC-conjugated anti-CD11a and PerCP-Cy55-conjugated anti-CCR9 (Biolegend)were added Stained cells were analyzed by five-color flowcytometry Lymphocytes were gated on the basis of theirforward- and side-scatter profiles Fluorescence data wererecorded and results are presented as percentages of adhesionmolecule- and chemokine receptor-expressing CD4+ andCD8+ lymphocytes Representative flow cytometry plots areshown in Figure 2(c)

26 RNA Extraction and Real-Time PCR Total RNA wasisolated from colon tissue using the Trizol reagent (Invi-trogen Carlsbad CA) RNA (1120583g) was reverse-transcribedwith a complementary (c)DNA synthesis kit (FermentasGlen Burnie MD USA) according to standard protocols Forreal-time PCR 5 120583L of 110 diluted cDNA was amplified in a25 120583L PCR volume containing 125 120583L of 2X SYBR greenmas-ter mix reagent (Applied Biosystems Foster City CA USA)The reaction was performed with ABI 7300 Real-Time PCRSystem (Applied Biosystems) according to the thermocycling

protocol recommended by the PCR system Primer sequenceswere as follows mouse ICAM-1 (51015840-AGCACCTCCCCA-CCTACTTT-31015840 and 51015840-AGCTTGCACGACCCTTCTAA-31015840) mouse P-selectin (51015840-TCCAGGAAGCTCTGACGT-ACTTG-31015840 and 51015840-GCAGCGTTAGTGAAGACTCCGTAT-31015840) mouse E-selectin (51015840-TGAACTGAAGGGATCAAG-AAGACT-31015840 and 51015840-GCCGAGGGACATCATCACAT-31015840)and mouse 18S rRNA (51015840-CGCGGTTCTATTTTGTTGGT31015840and 51015840-AGTCGGCATCGTTTATGGTC-31015840) All sampleswere analyzed in triplicate and fold change for each targetgene was calculated by the equation 2minusΔΔCt (ΔCt indicatesthe difference in threshold cycles between the test gene and18S rRNA and ΔΔCt indicates the difference of ΔCt betweenthe experimental and C groups)

27 Immunofluorescence Staining Double-staining combina-tions CD3-CD4 and CD3-CD8 were performed on 5 120583mparaffin-embedded colon sections After antigen retrievalsections were incubated with an antibody against CD3120576(Santa Cruz Biotechnology Santa Cruz CA USA) overnightat 4∘C and amplified with a rabbit anti-goat immunoglobulinG (IgG) secondary antibody conjugated with FITC (SantaCruz Biotechnology) For colocalization sections were thencostained overnight at 4∘Cwith secondary antibodies againstCD4 (Abcam Cambridge UK) or CD8 (Novus BiologicalsLittleton CO USA) and amplified with the respective appro-priate secondary antibodies goat anti-mouse IgG or goatanti-rabbit IgG conjugated with rhodamine (Santa CruzBiotechnology) Cell nuclei were counterstained with 410158406-diamidino-2-phenylindole (DAPI Sigma St Louis MOUSA) for 10min at room temperature Digital images at 400xmagnification per section were acquired using appropriatefilters of a Zeiss Axiophot fluorescence microscope (CarlZeiss MicroImaging LLC Thornwood NY USA) fitted witha Nikon D1X digital camera (Tokyo Japan) Cells containingboth FITC and rhodamine labels appeared yellow Theseimages were then overlaid with DAPI-staining images todetermine the infiltration of T lymphocyte subpopulations inthe colon mucosa

28 Statistical Analysis All data are expressed as the mean plusmnstandard error of themean (SEM) Differences among groupswere analyzed by an analysis of variance (ANOVA) withTukeyrsquos test A two-way ANOVA with Bonferroni correctionwas used to analyze differences in BW changes A 119875 value oflt005 was considered statistically significant

3 Results

31 BW and WeightLength Ratio of the Colon Initial BWsranged 21sim25 g and did not differ among the 4 groupsThere was no significant difference in BWs during the studybetween the C and G groups At 4 d (d 9) and 5 d (d 10) afterDSS administration weight loss was observed in the DCgroup compared to the C group whereas mice with Glnsupplementationmaintained their BWsduring theDSS expo-sure period At the end of the study BWs were significantlyhigher in the DG group than the DC group (Figure 3(a))

4 Mediators of Inflammation

81 80

F480

C

C

G

G

DC DG

DC DG

102

102

103

103

104

104

105

102

103

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102

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102

103

104

105

102

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102

103

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105

102

103

104

105

102

103

104

105

105

102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105

102 103 104 105 102 103 104 105 102 103 104 105

169 159

Ly6G Ly6G Ly6G Ly6G

CD19

CD3120576 CD3120576 CD3120576 CD3120576

Blood leukocytes

(a)

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105

C G DC DG

CD3120576 CD3120576 CD3120576 CD3120576

CD19

456 463 332 348

MLN cells

(b)

102 103 104 105

Gated cells

Gated cells

Gated cells

FSC

FSC

CD4 or CD8

PSGL-1 12057241205737

12057241205737

CD11a CCR9

CCR9

Cou

nt

Cou

nt

(c)

Figure 2 Representative flow cytometry plots Blood leukocytes (a) were defined by gating on CD45-positive cells The percentage of Ly6G-positive neutrophils from an individual representative mouse per group is listed For analyzing the lymphocyte population in MLNs (b)MLN cells were first gated to exclude debris Numbers indicate the percentage of CD3120576-positive lymphocytes in MLN cells For analyzingthe expression of adhesion molecules and chemokine receptors by T cells (c) lymphocytes were first identified based on low FSC and SSCcharacteristics CD4- or CD8-positive lymphocytes were gated to analyze the percentages of adhesion molecule- and chemokine receptor-expressing CD4+ and CD8+ lymphocytes Representative dot plots of leukocytes in blood are shown

Mediators of Inflammation 5

0 1 2 3 4 5 6 7 8 9 10

Body

wei

ght (

g)

0

18

20

22

24

26

28

30

CG

DC

Days

DG

daggerlowast

lowast

(a)

C G DC DG

Col

on w

eigh

tlen

gth

ratio

(gc

m)

000

002

004

006

008

010

daggerlowast

lowast

(b)

Figure 3 Body weight (a) and weightlength ratio of the colon (b) Data are presented as the mean plusmn SEM C normal mice fed the controldiet G normal mice fed a glutamine-enriched diet DC DSS group fed the control diet DG DSS group fed a glutamine-enriched dietlowastSignificantly different from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

The weightlength ratio of the colon an indicator of colonicedema was significantly higher in the colitis groups than theC group (Figure 3(b)) Treatment withGln attenuated colonicedema associated with DSS-induced inflammation

32 Leukocyte Populations in Blood and MLNs There wasno significant difference in blood or MLN leukocyte sub-populations between the C and G groups Compared to theC group the DSS colitis groups had a higher percentage ofblood neutrophils and lower T-cell population inMLNs DSSexposure did not alter the blood monocyte and lymphocytedistributions Also subsets of effector T cells in the blood andMLNs did not change Gln supplementation had no influenceon blood leukocyte or MLN lymphocyte populations innormal or colitic mice (Table 2)

33 Adhesion Molecule and CCR9 Expressions by Th CellsPercentages of adhesion molecules and CCR9 expressed byblood and MLN Th cells did not differ between the C and Ggroups DSS administration resulted in higher PSGL-1 CD11a(LFA-1 120572L subunit) and CCR9 expressions byThcells in bothblood and MLNs whereas no difference in 12057241205737 integrinexpression was detected among the control and DSS groupsMice in the DG group had lower percentages of PSGL-1-CD11a- and CCR9-expressing Th cells in blood (Figures4(a)ndash4(d)) and MLNs (Figures 5(a)ndash5(d)) The expressionlevel of CCR9 on 12057241205737-positive Th cells was also suppressedin the DG group (Figures 4(e) and 5(e))

34 Adhesion Molecule and CCR9 Expressions by Tc CellsNo differences in adhesion molecules and CCR9 expressedby blood and MLN Tc cells were observed between the Cand G groupsThe DSS colitis groups had higher percentages

Table 2 Leukocyte populations in blood and mesenteric lymphnodes (MLNs) ()

C G DC DGBlood

Neutrophils 87 plusmn 05 80 plusmn 09 161 plusmn 27lowast 169 plusmn 21lowast

Monocytes 72 plusmn 09 67 plusmn 07 82 plusmn 13 77 plusmn 15T cells 119 plusmn 04 117 plusmn 04 93 plusmn 12 93 plusmn 14

B cells 503 plusmn 09 534 plusmn 31 532 plusmn 27 490 plusmn 21

Th cells 67 plusmn 16 64 plusmn 04 73 plusmn 12 81 plusmn 08

Tc cells 36 plusmn 08 44 plusmn 03 45 plusmn 02 41 plusmn 03

MLNsT cells 452 plusmn 24 467 plusmn 22 321 plusmn 23

lowast

349 plusmn 10lowast

B cells 303 plusmn 18 290 plusmn 33 313 plusmn 21 297 plusmn 14

Th cells 118 plusmn 03 123 plusmn 07 112 plusmn 09 121 plusmn 09

Tc cells 63 plusmn 04 67 plusmn 06 58 plusmn 03 65 plusmn 05

CD45-positive cells were considered to be leukocytes and gated to determinethe population of leukocytes using a flow cytometer Staining for Ly-6G F480 CD3120576 and CD19 was used to respectively identify neutrophilmonocyte T cell and B cell populations For the analysis of T cell subpop-ulations lymphocytes were gated on the basis of their forward-and side-scatter profiles Percentages of T helper (Th) and cytotoxic T (Tc) cells wererespectively determined by CD4-and CD8-expressing cells in lymphocytesValues are presented as the mean plusmn SEM lowastSignificantly differs from the Cgroup (119875 lt 005)

of PSGL-1- CD11a- and CCR9-expressing blood and MLNTc cells whereas expression levels of 12057241205737 integrins did notdiffer among the 4 groups Compared to the DC group theDG group had lower expression of CD11a by blood (Figures6(a)ndash6(d)) and MLN Tc cells (Figures 7(a)ndash7(d)) There wasno difference in expression levels of CCR9 on 12057241205737-positiveTc cells between the 2 DSS colitis groups (Figures 6(e) and7(e))

6 Mediators of Inflammation

0

10

20

30

40

50

C G DC DG

PSG

L-1

()

lowast

lowast

dagger

(a)

0

2

4

6

8

10

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

5

10

15

20

25

C G DC DG

dagger

lowast

CD11

a (

)

(c)

0

10

20

30

40

50

C G DC DG

dagger

lowast

lowastCCR9

(

)

(d)

0

15

30

45

60

C G DC DG

lowast

CCR

9 ex

pres

sing

dagger

CD4+12057241205737+

T ce

lls (

)

(e)

Figure 4 Percentage of adhesionmolecule- and chemokine receptor-expressingThelper (Th) cells in blood CD4-positive blood lymphocyteswere gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d)) (e) Expression of CCR9 by CD4and 12057241205737 integrin double-positive blood lymphocytes Values are shown as the mean plusmn SEM lowastSignificantly different from the C group(119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

35 Gene Expression of Endothelial Adhesion Molecules inColon Tissues There was no difference in mRNA levels ofICAM-1 P-selectin and E-selectin between the C and Ggroups in colon tissues DSS-induced colitis greatly upreg-ulated the adhesion molecule genes expressed by activatedendothelial cells Compared to the DC group the expression

levels of ICAM-1 P-selectin and E-selectin mRNA weresuppressed in the DG group (Figure 8)

36 T Lymphocyte Subsets in the Colon Mucosa CD3 is thecell surface marker of T cells CD3 and CD4 double-positivecells are considered Th cells whereas Tc cells coexpress CD3

Mediators of Inflammation 7

0

10

20

30

40

50

C G DC DG

PSG

L-1

()

lowast

lowast

dagger

(a)

0

2

4

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8

10

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

10

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C G DC DG

CD11

a (

)

dagger

lowast

(c)

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C G DC DG

dagger

lowast

CCR9

(

)

(d)

0

15

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45

60

C G DC DG

dagger

lowast

CCR

9 ex

pres

sing

CD4+12057241205737+

T ce

lls (

)

(e)

Figure 5 Percentage of adhesion molecule- and chemokine receptor-expressing T helper (Th) cells in mesenteric lymph nodes (MLNs)CD4-positiveMLN lymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d))(e) Expression of CCR9 by CD4 and 12057241205737 integrin double-positive MLN lymphocytes Values are shown as the mean plusmn SEM lowastSignificantlydifferent from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

andCD8As shown in Figure 9 the immunoreactive intensityof Th cells was higher in the DC group than the DG groupHowever intensities of Tc cell populations did not differbetween the DC and DG groups (Figure 10)

4 Discussion

DSS is a heparin-like polysaccharide which results in acutechemical toxicity that disrupts the intestinal epithelial cell

8 Mediators of Inflammation

0

10

20

30

40

50

C G DC DG

lowast lowast

PSG

L-1

()

(a)

0

5

10

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20

25

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

5

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C G DC DG

dagger

lowast

CD11

a (

)

(c)

0

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C G DC DG

CCR9

()

lowast

lowast

(d)

0

20

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100

C G DC DG

lowastlowast

CCR

9 ex

pres

sing

CD8+12057241205737+

T ce

lls (

)

(e)

Figure 6 Percentage of adhesion molecule- and chemokine receptor-expressing cytotoxic T (Tc) cells in blood CD8-positive bloodlymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d)) (e) Expressionof CCR9 by CD8 and 12057241205737 integrin double-positive blood lymphocytes Values are shown as the mean plusmn SEM lowastSignificantly different fromthe C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

barrier [21] DSS-induced colitis is characterized by extensivecrypt and epithelial cell damage with ulceration tissueedema and infiltration of immune cells predominantly in thedistal colon that mimics the histological features of UC [22]Recent studies indicated that DSS-induced morphological

and biochemical damage also extends to the small intestines[23] Susceptibilities to DSS-induced colitis differ in variousinbred mouse strains [24] A single cycle of DSS exposureto the C57BL6 strain was found to develop acute colitiswhich later proceeds to chronic inflammation and T cell

Mediators of Inflammation 9

C G DC DG

PSG

L-1

()

0

10

20

30

40

50

lowastlowast

(a)

CD11

a (

)

C G DC DG0

5

10

15

20

25

CD11

a (

)

12057241205737

inte

grin

s (

)

(b)

C G DC DG0

5

10

15

20

25

CD11

a (

)

dagger

lowast

(c)

CCR9

()

C G DC DG0

5

10

15

20

25

lowast

lowast

(d)

CCR

9 ex

pres

sing

0

15

30

45

60

lowastlowast

CD+ 812057241205737+

T ce

lls (

)

C G DC DG

(e)

Figure 7 Percentage of adhesion molecule- and chemokine receptor-expressing cytotoxic T (Tc) cells in mesenteric lymph nodes (MLNs)CD8-positiveMLN lymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d))(e) Expression of CCR9 by CD8 and 12057241205737 integrin double-positive MLN lymphocytes Values are shown as the mean plusmn SEM lowastSignificantlydifferent from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

migration to the colon plays an important role in theprogression to chronicity [25] Regarding the high sensitivityto DSS C57BL6 mice were used in this study to analyze theconsequences of DSS exposure on adhesion molecules and

chemokine receptors involved in T cell trafficking to theintestines

IBD is associated with a massive influx of immune cellsinto the gut Previous studies indicated that increased local

10 Mediators of Inflammation

ICAM-1 P-selectin E-selectin

Relat

ive m

RNA

ratio

0

50

100

150

200

1000

1250

1500

CG

DCDG

daggerdagger

dagger

lowast

lowastlowast

lowast

lowast

lowast

Figure 8 Gene expression of endothelial adhesion molecules incolon tissues mRNA levels were analyzed by a real-time PCR TheC group was used as a calibrator and the data were presented as thefold change in gene expression relative to the calibrator Values areshown as themean plusmn SEM lowastSignificantly different from the C group(119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

secretion of proinflammatory cytokines by the inflamedcolon leads to upregulated expressions of vascular adhesionmolecules resulting in a sustained influx of inflammatorycells [26] Although inflammation predominantly occurs inthe GI tract IBD patients are likely to develop extraintestinalmanifestations [27] that may be attributed to aberrant acti-vation and homing of T cells [28] A recent study indicatedthat circulating CD4+ and CD8+ T cells are activated in bothCD and UC patients and these cells were correlated withleakage of microbial products from the impaired intestinalbarrier [5] Also the severity of DSS-induced colitis wascorrelatedwith the immune response ofMLNs [29] A similarphenomenon was also observed in this study Our resultsindicated that expressions of adhesion molecules and CCR9on Th cells and Tc cells significantly increased in both bloodand MLNs after DSS exposure These findings suggest thatboth local and systemic T cells are activated The role of Thcells in the pathogenesis of IBD has been widely studiedDysregulation ofTh cells can lead to immune cell infiltrationinto the intestinal mucosa and cause persistent inflammation[3 6] The pathogenic role of Tc cells in IBD has been lessinvestigated A previous study showed that antigen-specificTc cells caused relapsing colitis in normal mice due to thecytolytic function against the intestinal epithelium [30] Leeet al [4] reported a gene expression profile of circulatingCD8+ T cells that predicted a more aggressive disease coursefor IBD patients However the modulatory mechanism of Tccells in IBD is still under investigation

Naıve T cells constantly recirculate between the bloodand secondary lymphoid organs Once activated in secondarylymphoid organs they become effector T cells that expressadhesion molecules and chemokine receptors which controltheir extravasation into nonlymphoid tissue sites [31] T cell

trafficking to the gut and gut-associated lymphoid tissues(GALTs) requires 12057241205737 integrins The ligand MAdCAM-1is constitutively expressed by the mucosal endothelium inthe small intestine and colon [32] CCR9 is thought toparticipate in the specific localization of T cells to the smallintestines because the sole ligand for CCR9 C-C chemokineligand 25 (CCL25) is strongly expressed by the small intesti-nal epithelium [33] However Wurbel et al [34] revealedthat CCL25 expression increased during the recovery phaseafter acute DSS administration suggesting a regulatory roleof CCR9CCL25 interactions during colonic inflammationIn this study percentages of PSGL-1- LFA-1- and CCR9-expressing T cells were upregulated in acute DSS colitiswhereas expression levels of 12057241205737 integrins in colitic micedid not differ from those of normal mice It is possible thatT cell trafficking into the gut during acute DSS exposure isless dependent on 12057241205737 integrins In support of our findingsWang et al [35] reported that localization of T cells to theintestines was relatively unaffected by 12057241205737 blockade duringacute DSS-induced colitis

Gln is a critical fuel source for enterocytes and immunecells Gln supplementation attenuates gut injury by a com-plex mechanism which involves protecting the epithelialbarrier function reducing oxidative stress and modulat-ing inflammatory responses [36] The local and systemicimmunomodulatory effects of Gln have been discussed invarious experimental colitis models via different adminis-tration routes Studies using rodents with trinitrobenzenesulfonic acid-induced colitis indicated that Gln given bythe rectal route inhibits nuclear factor (NF)-120581B- and STAT-mediated inflammation in colon tissues [15] and further pre-vents colon fibrosis through downregulating gene pathwaysthat contribute to the accumulation of matrix proteins [37]We recently demonstrated that intraperitoneal pretreatmentwith alanyl-Gln a Gln-containing dipeptide widely used inparenteral nutrition suppresses cytokine expression in bloodTh cells reduces NF-120581B-mediated inflammatory responsesin the colon and upregulates expressions of genes whichpromote recovery of the colonic mucosa [17 38]

Because most exogenous Gln is absorbed in the proximalsmall intestine it might not reach the inflamed colon at asufficient concentration tomodulate inflammatory responses[36] However the enteral route of Gln administrationstill showed protective effects against DSS-induced damageOral Gln supplementation reduced the feces water contentenhanced expression of HSPs in the colonic mucosa andameliorated colon injury caused byDSS exposure [16 39 40]A previous study indicated that oral Gln attenuated leukocyteadhesion and emigration in a rodentmodel of indomethacin-induced ileitis [41] In this study we demonstrated thatoral Gln administration suppressed adhesion molecules andCCR9 expressed by T cells and downregulated the mRNAlevels of adhesion molecules expressed by endothelium incolon tissues The histological findings also support theresults that Gln administration suppressed the infiltrationof Th cells into the colon mucosa Gln consumption is animportant component of T cell activation [42 43] Differentsusceptibilities ofThandTc cells toGln supplementationmaybe explained by a higher cell population of Th cells which

Mediators of Inflammation 11

50120583m

50120583m

50120583m

50120583m

CD3

C

G

DC

DG

(a)

50120583m

50120583m

50120583m

50120583m

CD4

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 9 Representative examples of the immunofluorescence staining of T helper cells In the left column cell surface antigens were stainedfor CD3 (green FITC) and staining of CD4 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI(blue) and the last column presents a colocalized fluorescence image of CD3+CD4+ T cells

competes as a Gln source with Tc cells Further studies areneeded to investigate the molecular mechanisms involved ingene expressions of adhesion molecules and CCR9 regulatedby Gln

In conclusion this study showed for the first time thatpretreatment with oral Gln reduced adhesion molecule- andCCR9-expressing T cells induced by DSS exposure Theinhibitory abilities against adhesion molecule and CCR9expressions were more obvious inTh cells than Tc cells AlsoGln supplementation reduced gene expressions of endothe-lial adhesion molecules in colons prevented BW loss and

attenuated colon edema in coliticmice Our results imply thatdietary Gln prevented Th cell trafficking into colon tissuesand provide a new mechanism of Gln supplementation thathas beneficial effects on ameliorating the severity of acuteDSS-induced colitis

Conflict of Interests

The authors declare that they have no conflict of interests

12 Mediators of Inflammation

50120583m

CD3

50120583m

50120583m

50120583m

C

G

DC

DG

(a)

CD8

50120583m

50120583m

50120583m

50120583m

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 10 Representative immunofluorescence images of cytotoxic T cells In the left column cell surface antigens were stained for CD3(green FITC) and the staining of CD8 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI (blue)and the last column presents a colocalized fluorescence image of CD3+CD8+ T cells

Acknowledgments

The authors would like to thank Man-Hui Pai Assistant Pro-fessor of Department of Anatomy Taipei Medical Universityfor her technical assistance in immunofluorescence stainingThis studywas supported by aResearchGrant (NSC100-2320-B-038-007-MY3) from the National Science Council TaipeiTaiwan

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] C T Murphy K Nally F Shanahan and S Melgar ldquoShining alight on intestinal trafficrdquoClinical and Developmental Immunol-ogy vol 2012 Article ID 808157 14 pages 2012

Mediators of Inflammation 13

[3] L A Zenewicz A Antov and R A Flavell ldquoCD4 T-cell differ-entiation and inflammatory bowel diseaserdquo Trends inMolecularMedicine vol 15 no 5 pp 199ndash207 2009

[4] J C Lee P A Lyons E F McKinney et al ldquoGene expressionprofiling of CD8+ T cells predicts prognosis in patients withCrohn disease and ulcerative colitisrdquo Journal of Clinical Inves-tigation vol 121 no 10 pp 4170ndash4179 2011

[5] N T Funderburg S R Stubblefield Park H C Sung et alldquoCirculating CD4+ and CD8+ T cells are activated in IBDand are associated with plasma markers of inflammationrdquoImmunology vol 140 no 1 pp 87ndash97 2013

[6] E J Villablanca B Cassani U H von Andrian and J RMora ldquoBlocking lymphocyte localization to the gastrointestinalmucosa as a therapeutic strategy for inflammatory boweldiseasesrdquo Gastroenterology vol 140 no 6 pp 1776ndash1784 2011

[7] R P McEver and R D Cummings ldquoPerspectives series celladhesion in vascular biology Role of PSGL-1 binding toselectins in leukocyte recruitmentrdquo The Journal of ClinicalInvestigation vol 100 no 3 pp 485ndash491 1997

[8] C Berlin E L Berg M J Briskin et al ldquo12057241205737 Integrin medi-ates lymphocyte binding to the mucosal vascular addressinMAdCAM-1rdquo Cell vol 74 no 1 pp 185ndash195 1993

[9] K Ley C Laudanna M I Cybulsky and S Nourshargh ldquoGet-ting to the site of inflammation the leukocyte adhesion cascadeupdatedrdquoNature Reviews Immunology vol 7 no 9 pp 678ndash6892007

[10] B Moser M Wolf A Walz and P Loetscher ldquoChemokinesmultiple levels of leukocyte migration controlrdquo Trends inImmunology vol 25 no 2 pp 75ndash84 2004

[11] S Arimilli W Ferlin N Solvason S Deshpande M Howardand S Mocci ldquoChemokines in autoimmune diseasesrdquo Immuno-logical Reviews vol 177 pp 43ndash51 2000

[12] C Koenecke and R Forster ldquoCCR9 and inflammatory boweldiseaserdquo Expert Opinion on Therapeutic Targets vol 13 no 3pp 297ndash306 2009

[13] B Johansson-Lindbom and W W Agace ldquoGeneration of gut-homing T cells and their localization to the small intestinalmucosardquo Immunological Reviews vol 215 no 1 pp 226ndash2422007

[14] P EWischmeyer ldquoGlutaminemode of action in critical illnessrdquoCritical Care Medicine vol 35 no 9 pp S541ndashS544 2007

[15] N A Kretzmann H Fillmann J L Mauriz C A MarroniJ Gonzalez-Gallego and M J Tunon ldquoEffects of glutamineon proinflammatory gene expression and activation of nuclearfactor Kappa B and signal transducers and activators oftranscription in TNBS-induced colitisrdquo Inflammatory BowelDiseases vol 14 no 11 pp 1504ndash1513 2008

[16] H Xue A J D Sufit and P EWischmeyer ldquoGlutamine therapyimproves outcome of in vitro and in vivo experimental colitismodelsrdquo Journal of Parenteral and Enteral Nutrition vol 35 no2 pp 188ndash197 2011

[17] C-C Chu Y-C Hou M-H Pai C-J Chao and S-L YehldquoPretreatment with alanyl-glutamine suppresses T-helper-cell-associated cytokine expression and reduces inflammatoryresponses in mice with acute DSS-induced colitisrdquo The Journalof Nutritional Biochemistry vol 23 no 9 pp 1092ndash1099 2011

[18] S Kew S M Wells P Yaqoob F A Wallace E A Miles and PC Calder ldquoDietary glutamine enhances murine T-lymphocyteresponsivenessrdquo The Journal of Nutrition vol 129 no 8 pp1524ndash1531 1999

[19] S M Wells S Kew P Yaqoob F A Wallace and P C CalderldquoDietary glutamine enhances cytokine production by murinemacrophagesrdquo Nutrition vol 15 no 11-12 pp 881ndash884 1999

[20] Y-C Hou M-H Pai W-C Chiu Y-M Hu and S-L YehldquoEffects of dietary glutamine supplementation on lung injuryinduced by lipopolysaccharide administrationrdquo American Jour-nal of PhysiologymdashLung Cellular and Molecular Physiology vol296 no 3 pp L288ndashL295 2009

[21] M Kawada A Arihiro and E Mizoguchi ldquoInsights fromadvances in research of chemically induced experimental mod-els of human inflammatory bowel diseaserdquo World Journal ofGastroenterology vol 13 no 42 pp 5581ndash5593 2007

[22] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[23] R Yazbeck G S Howarth R N Butler M S Geier and CA Abbott ldquoBiochemical and histological changes in the smallintestine of mice with dextran sulfate sodium colitisrdquo Journal ofCellular Physiology vol 226 no 12 pp 3219ndash3224 2011

[24] M Mahler I J Bristol E H Leiter et al ldquoDifferentialsusceptibility of inbred mouse strains to dextran sulfatesodium-induced colitisrdquo American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 274 no 3 part 1 ppG544ndashG551 1998

[25] S Melgar A Karlsson and E Michaelsson ldquoAcute colitisinduced by dextran sulfate sodium progresses to chronicityin C57BL6 but not in BALBc mice correlation betweensymptoms and inflammationrdquoAmerican Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 288 no 6 pp G1328ndashG1338 2005

[26] MKoizumiN King R Lobb C Benjamin andDK PodolskyldquoExpression of vascular adhesion molecules in inflammatorybowel diseaserdquo Gastroenterology vol 103 no 3 pp 840ndash8471992

[27] L Peyrin-Biroulet E V Loftus Jr J-F Colombel and W JSandborn ldquoLong-term complications extraintestinal manifes-tations and mortality in adult Crohnrsquos disease in population-based cohortsrdquo Inflammatory Bowel Diseases vol 17 no 1 pp471ndash478 2011

[28] D H Adams and B Eksteen ldquoAberrant homing of mucosal Tcells and extra-intestinal manifestations of inflammatory boweldiseaserdquoNature Reviews Immunology vol 6 no 3 pp 244ndash2512006

[29] TW Spahn H Herbst P D Rennert et al ldquoInduction of colitisinmice deficient of Peyerrsquos patches andmesenteric lymph nodesis associated with increased disease severity and formation ofcolonic lymphoid patchesrdquo The American Journal of Pathologyvol 161 no 6 pp 2273ndash2282 2002

[30] S Nancey S Holvoet I Graber et al ldquoCD8+ Cytotoxic T CellsInduce Relapsing Colitis in Normal Micerdquo Gastroenterologyvol 131 no 2 pp 485ndash496 2006

[31] E C Butcher and L J Picker ldquoLymphocyte homing and home-ostasisrdquo Science vol 272 no 5258 pp 60ndash66 1996

[32] C Berlin R F Bargatze J J Campbell et al ldquo1205724 Integrinsmediate lymphocyte attachment and rolling under physiologicflowrdquo Cell vol 80 no 3 pp 413ndash422 1995

[33] K A Papadakis J Prehn V Nelson et al ldquoThe role of thymus-expressed chemokine and its receptor CCR9 on lymphocytesin the regional specialization of the mucosal immune systemrdquoJournal of Immunology vol 165 no 9 pp 5069ndash5076 2000

14 Mediators of Inflammation

[34] M-A Wurbel M G McIntire P Dwyer and E FiebigerldquoCCL25CCR9 interactions regulate large intestinal inflamma-tion in a murine model of acute colitisrdquo PLoS ONE vol 6 no 1Article ID e16442 2011

[35] CWang E K Hanly LWWheeler M Kaur K GMcDonaldand R D Newberry ldquoEffect of 12057241205737 blockade on intestinallymphocyte subsets and lymphoid tissue developmentrdquo Inflam-matory Bowel Diseases vol 16 no 10 pp 1751ndash1762 2010

[36] M Coeffier R Marion-Letellier and P Dechelotte ldquoPotentialfor amino acids supplementation during inflammatory boweldiseasesrdquo Inflammatory Bowel Diseases vol 16 no 3 pp 518ndash524 2010

[37] B San-Miguel I Crespo N A Kretzmann et al ldquoGlutamineprevents fibrosis development in rats with colitis induced by246-trinitrobenzene sulfonic acidrdquo The Journal of Nutritionvol 140 no 6 pp 1065ndash1071 2010

[38] Y C Hou C C Chu T L Ko et al ldquoEffects of alanyl-glutaminedipeptide on the expression of colon-inflammatory mediatorsduring the recovery phase of colitis induced by dextran sulfatesodiumrdquo European Journal of Nutrition vol 52 no 3 pp 1089ndash1098 2013

[39] Y Shiomi S Nishiumi M Ooi et al ldquoGCMS-based meta-bolomic study in mice with colitis induced by dextran sulfatesodiumrdquo Inflammatory Bowel Diseases vol 17 no 11 pp 2261ndash2274 2011

[40] M Vicario C Amat M Rivero M Moreto and C PelegrıldquoDietary glutamine affects mucosal functions in rats with mildDSS-induced colitisrdquoThe Journal of Nutrition vol 137 no 8 pp1931ndash1937 2007

[41] H Arndt F Kullmann F Reuszlig J Scholmerich and K-D Pal-itzsch ldquoGlutamine attenuates leukocyte-endothelial cell adhe-sion in indomethacin-induced intestinal inflammation in theratrdquo Journal of Parenteral and Enteral Nutrition vol 23 no 1pp 12ndash18 1999

[42] P Yaqoob and P C Calder ldquoGlutamine requirement of prolif-erating T lymphocytesrdquo Nutrition vol 13 no 7-8 pp 646ndash6511997

[43] E L Carr A Kelman G S Wu et al ldquoGlutamine uptake andmetabolism are coordinately regulated by ERKMAPK duringT lymphocyte activationrdquo Journal of Immunology vol 185 no2 pp 1037ndash1044 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Mediators of Inflammation

Chemokines are small peptides which bind to chemokinereceptors expressed on leukocytes and function as chemoat-tractants They regulate lymphocyte homing to secondarylymphoid organs and transmigration into tissues by forminga chemokine concentration gradient which attracts lympho-cytes to move towards an increasing concentration [10]Dysregulation of chemokines and chemokine receptors wasimplicated in various autoimmune diseases including IBD[11 12] Recent studies indicated that C-C chemokine receptortype 9 (CCR9) and 12057241205737 integrins are required for thelocalization of lymphocytes to the GI mucosa [13] Agentsdeveloped for disrupting actions of CCR9 and 12057241205737 integrinsshowed promising results in IBD clinical trials [6]

Glutamine (Gln) is an immunomodulatory nutrientwhich is widely used in clinical practice [14] Previous studiesshowed that Gln treatment has beneficial effects in differentexperimental models of colitis Gln attenuates the expressionof proinflammatory mediators [15] and improves outcomeswhich may be due to upregulation of heat shock proteins(HSPs) [16] Recent work in our laboratory demonstratedthat pretreatment with Gln suppresses cytokine expression ofTh cells and ameliorates the severity of acute dextran sulfatesodium- (DSS-) induced colitis [17] However whether thebeneficial effects of Gln are mediated by modulating lym-phocyte trafficking in colitis is still unclear Therefore weinvestigated the influence of dietary Gln supplementation onT cell adhesion molecules and CCR9 expression in mice withDSS-induced acute colitis

2 Materials and Methods

21 Study Protocols Six-week-old male C57BL6 mice wereused in this study Conventional mice were maintained ina temperature- and humidity-controlled room and were feda standard chow diet ad libitum before the study Care oflaboratory animals was in full compliance with the Guide forthe Care and Use of Laboratory Animals (National ResearchCouncil 1996) and protocols were approved by the Institu-tional Animal Care and Use Committee of Taipei MedicalUniversity

22 Experimental Design After 1 week of acclimation 40mice were randomly assigned to a control group or a Glngroup in this study with 20 mice in each group Mice in thecontrol group were fed a common semipurified diet whilethe Gln group received a diet in which part of the caseinwas replaced with Gln Gln provided 25 of the total aminoacid nitrogen This amount of Gln was proven to have animmunomodulatory effect in rodents [18ndash20] Two diets wereformulated to be isonitrogenous and isoenergetic (Table 1)After 5 d of being fed the diets mice in the control and Glngroups were further divided into 2 respective subgroups Onesubgroup was given distilled water while the other subgroupreceived 15 (wtvol) DSS (MW40 kDa MP BiomedicalsSolon OH USA) in the drinking water for 5 d to inducecolitis A flow diagram of the study design is shown inFigure 1 There were 4 groups in this study control diet withdistilled water (C group) Gln diet with distilled water (Ggroup) control diet with DSS water (DC group) and Gln

Table 1 Composition of the semipurified diets

Component Control diet Gln dietgkg

Soybean oil 100 100Casein 200 150Glutamine 0 417Salt mixturea 35 35Vitamin mixtureb 10 10Methyl cellulose 31 31Choline bitartrate 25 25Methionine 3 3Corn starch 6268 6185aThe salt mixture contained the following (mgg) calcium phosphatediabasic 500 sodium chloride 74 potassium sulfate 52 potassium citratemonohydrate 20 magnesium oxide 24 manganese carbonate 35 ferriccitrate 6 zinc carbonate 16 curpric carbonate 03 potassium iodate 001sodium selenite 001 and chromium potassium sulfate 055bThe vitamin mixture contained the following (mgg) thiamin hydrochlo-ride 06 riboflavin 06 pyridoxine hydrochloride 07 nicotinic acid 3calcium pantothenate 16 D-biotin 005 cyanocobalamin 0001 retinylpalmitate 16 DL-120572-tocopherol acetate 20 cholecalciferol 025 andmenaquinone 0005

Sacrifice(days)

Induction of

Water

DCDG

Con dietColitis groups

C Con dietGln diet

Gln diet

G

Healthygroups Water

15 DSS15 DSS

D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 D10

colitis (n = 10per group)

control

Figure 1 Flow diagram of the study design

diet withDSSwater (DG group)The respective experimentaldiets were given during the DSS exposure period Bodyweights (BWs) were recorded daily and all mice had freeaccess to food and water throughout the study At the endof the experiment mice were anesthetized and sacrificed bycardiac puncture Fresh blood samples were collected inheparinized tubes for measurements of the leukocyte popu-lation Mesenteric lymph nodes (MLNs) were removed andprocessed for further analysis by flow cytometry The colonwas cut close to the ileocecal valve and its length and weightwere measured Sections (1 cm) of the distal colon were cutColon tissues were fixed with buffered 4 paraformaldehydefor an immunohistochemical analysis

23 Blood Leukocyte Distribution A five-color flow cyto-metric analysis was performed to determine the distributionof peripheral blood leukocytes Antibodies against mouseleukocyte surface antigens were added to 100 120583L aliquots ofwhole blood The antibodies used to detect different sub-sets of leukocytes were as follows PerCP-conjugated anti-CD45 (Biolegend San Diego CA USA) for leukocytes PE-conjugated anti-F480 (eBioscience SanDiego CA USA) for

Mediators of Inflammation 3

monocytesmacrophages FITC-conjugated anti-Ly6G (BDBiosciences San Jose CA USA) for neutrophils APC-con-jugated anti-CD3120576 (eBioscience) for T cells and Pacific blue-conjugated anti-CD19 (Biolegend) for B cells Antibodieswere used at the concentration recommended by manufac-turer After a 30min incubation at 4∘C in the dark red bloodcells were lysed and cells were suspended in staining bufferand then analyzed with a FACS Canto II flow cytometer (BDBiosciences) CD45-positive cells were gated and results arepresented as a percentage of specific CD-marker-expressingcells in blood leukocytes Representative flow cytometry plotsare shown in Figure 2(a)

24 Lymphocyte Populations inMLNs Cell suspensions fromMLNs were obtained by passing the tissues through anylon cell strainer with a 40 120583m pore size (BD Biosciences)in RPMI1640 medium (Biological Industries Kibbutz BeitHaemek Israel) After centrifugation at 300timesg for 10minpelletedMLN cells were suspended in 1mL of staining bufferOne hundred microliters of cell suspension was incubatedwith APC-conjugated anti-CD3120576 (eBioscience) and Pacificblue-conjugated anti-CD19 (Biolegend) for 30min at 4∘Cin the dark Stained cells were washed and resuspended instaining buffer to measure the lymphocyte population byflow cytometry Percentages of T and B lymphocytes weredetermined by CD3120576- and CD19-expressing cells in MLNcells Representative flow cytometry plots are shown inFigure 2(b)

25 Expressions of Adhesion Molecules and Chemokine Recep-tors by T Cells Whole blood and MLNs were used to ana-lyze adhesion molecule- and chemokine receptor-express-ing T cells Whole blood and MLN cells obtained asdescribed above were split into 2 vials with 100 120583L ineach aliquot and these were incubated with Pacific blue-conjugated anti-CD4 (BD Biosciences) or Pacific blue-conjugated anti-CD8 antibodies (Biolegend) To investigateexpressions of adhesion molecules and chemokine receptorsPE-conjugated anti-PSGL-1 antibodies (BD Biosciences)APC-conjugated anti-12057241205737 integrin FITC-conjugated anti-CD11a and PerCP-Cy55-conjugated anti-CCR9 (Biolegend)were added Stained cells were analyzed by five-color flowcytometry Lymphocytes were gated on the basis of theirforward- and side-scatter profiles Fluorescence data wererecorded and results are presented as percentages of adhesionmolecule- and chemokine receptor-expressing CD4+ andCD8+ lymphocytes Representative flow cytometry plots areshown in Figure 2(c)

26 RNA Extraction and Real-Time PCR Total RNA wasisolated from colon tissue using the Trizol reagent (Invi-trogen Carlsbad CA) RNA (1120583g) was reverse-transcribedwith a complementary (c)DNA synthesis kit (FermentasGlen Burnie MD USA) according to standard protocols Forreal-time PCR 5 120583L of 110 diluted cDNA was amplified in a25 120583L PCR volume containing 125 120583L of 2X SYBR greenmas-ter mix reagent (Applied Biosystems Foster City CA USA)The reaction was performed with ABI 7300 Real-Time PCRSystem (Applied Biosystems) according to the thermocycling

protocol recommended by the PCR system Primer sequenceswere as follows mouse ICAM-1 (51015840-AGCACCTCCCCA-CCTACTTT-31015840 and 51015840-AGCTTGCACGACCCTTCTAA-31015840) mouse P-selectin (51015840-TCCAGGAAGCTCTGACGT-ACTTG-31015840 and 51015840-GCAGCGTTAGTGAAGACTCCGTAT-31015840) mouse E-selectin (51015840-TGAACTGAAGGGATCAAG-AAGACT-31015840 and 51015840-GCCGAGGGACATCATCACAT-31015840)and mouse 18S rRNA (51015840-CGCGGTTCTATTTTGTTGGT31015840and 51015840-AGTCGGCATCGTTTATGGTC-31015840) All sampleswere analyzed in triplicate and fold change for each targetgene was calculated by the equation 2minusΔΔCt (ΔCt indicatesthe difference in threshold cycles between the test gene and18S rRNA and ΔΔCt indicates the difference of ΔCt betweenthe experimental and C groups)

27 Immunofluorescence Staining Double-staining combina-tions CD3-CD4 and CD3-CD8 were performed on 5 120583mparaffin-embedded colon sections After antigen retrievalsections were incubated with an antibody against CD3120576(Santa Cruz Biotechnology Santa Cruz CA USA) overnightat 4∘C and amplified with a rabbit anti-goat immunoglobulinG (IgG) secondary antibody conjugated with FITC (SantaCruz Biotechnology) For colocalization sections were thencostained overnight at 4∘Cwith secondary antibodies againstCD4 (Abcam Cambridge UK) or CD8 (Novus BiologicalsLittleton CO USA) and amplified with the respective appro-priate secondary antibodies goat anti-mouse IgG or goatanti-rabbit IgG conjugated with rhodamine (Santa CruzBiotechnology) Cell nuclei were counterstained with 410158406-diamidino-2-phenylindole (DAPI Sigma St Louis MOUSA) for 10min at room temperature Digital images at 400xmagnification per section were acquired using appropriatefilters of a Zeiss Axiophot fluorescence microscope (CarlZeiss MicroImaging LLC Thornwood NY USA) fitted witha Nikon D1X digital camera (Tokyo Japan) Cells containingboth FITC and rhodamine labels appeared yellow Theseimages were then overlaid with DAPI-staining images todetermine the infiltration of T lymphocyte subpopulations inthe colon mucosa

28 Statistical Analysis All data are expressed as the mean plusmnstandard error of themean (SEM) Differences among groupswere analyzed by an analysis of variance (ANOVA) withTukeyrsquos test A two-way ANOVA with Bonferroni correctionwas used to analyze differences in BW changes A 119875 value oflt005 was considered statistically significant

3 Results

31 BW and WeightLength Ratio of the Colon Initial BWsranged 21sim25 g and did not differ among the 4 groupsThere was no significant difference in BWs during the studybetween the C and G groups At 4 d (d 9) and 5 d (d 10) afterDSS administration weight loss was observed in the DCgroup compared to the C group whereas mice with Glnsupplementationmaintained their BWsduring theDSS expo-sure period At the end of the study BWs were significantlyhigher in the DG group than the DC group (Figure 3(a))

4 Mediators of Inflammation

81 80

F480

C

C

G

G

DC DG

DC DG

102

102

103

103

104

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

105

102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105

102 103 104 105 102 103 104 105 102 103 104 105

169 159

Ly6G Ly6G Ly6G Ly6G

CD19

CD3120576 CD3120576 CD3120576 CD3120576

Blood leukocytes

(a)

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105

C G DC DG

CD3120576 CD3120576 CD3120576 CD3120576

CD19

456 463 332 348

MLN cells

(b)

102 103 104 105

Gated cells

Gated cells

Gated cells

FSC

FSC

CD4 or CD8

PSGL-1 12057241205737

12057241205737

CD11a CCR9

CCR9

Cou

nt

Cou

nt

(c)

Figure 2 Representative flow cytometry plots Blood leukocytes (a) were defined by gating on CD45-positive cells The percentage of Ly6G-positive neutrophils from an individual representative mouse per group is listed For analyzing the lymphocyte population in MLNs (b)MLN cells were first gated to exclude debris Numbers indicate the percentage of CD3120576-positive lymphocytes in MLN cells For analyzingthe expression of adhesion molecules and chemokine receptors by T cells (c) lymphocytes were first identified based on low FSC and SSCcharacteristics CD4- or CD8-positive lymphocytes were gated to analyze the percentages of adhesion molecule- and chemokine receptor-expressing CD4+ and CD8+ lymphocytes Representative dot plots of leukocytes in blood are shown

Mediators of Inflammation 5

0 1 2 3 4 5 6 7 8 9 10

Body

wei

ght (

g)

0

18

20

22

24

26

28

30

CG

DC

Days

DG

daggerlowast

lowast

(a)

C G DC DG

Col

on w

eigh

tlen

gth

ratio

(gc

m)

000

002

004

006

008

010

daggerlowast

lowast

(b)

Figure 3 Body weight (a) and weightlength ratio of the colon (b) Data are presented as the mean plusmn SEM C normal mice fed the controldiet G normal mice fed a glutamine-enriched diet DC DSS group fed the control diet DG DSS group fed a glutamine-enriched dietlowastSignificantly different from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

The weightlength ratio of the colon an indicator of colonicedema was significantly higher in the colitis groups than theC group (Figure 3(b)) Treatment withGln attenuated colonicedema associated with DSS-induced inflammation

32 Leukocyte Populations in Blood and MLNs There wasno significant difference in blood or MLN leukocyte sub-populations between the C and G groups Compared to theC group the DSS colitis groups had a higher percentage ofblood neutrophils and lower T-cell population inMLNs DSSexposure did not alter the blood monocyte and lymphocytedistributions Also subsets of effector T cells in the blood andMLNs did not change Gln supplementation had no influenceon blood leukocyte or MLN lymphocyte populations innormal or colitic mice (Table 2)

33 Adhesion Molecule and CCR9 Expressions by Th CellsPercentages of adhesion molecules and CCR9 expressed byblood and MLN Th cells did not differ between the C and Ggroups DSS administration resulted in higher PSGL-1 CD11a(LFA-1 120572L subunit) and CCR9 expressions byThcells in bothblood and MLNs whereas no difference in 12057241205737 integrinexpression was detected among the control and DSS groupsMice in the DG group had lower percentages of PSGL-1-CD11a- and CCR9-expressing Th cells in blood (Figures4(a)ndash4(d)) and MLNs (Figures 5(a)ndash5(d)) The expressionlevel of CCR9 on 12057241205737-positive Th cells was also suppressedin the DG group (Figures 4(e) and 5(e))

34 Adhesion Molecule and CCR9 Expressions by Tc CellsNo differences in adhesion molecules and CCR9 expressedby blood and MLN Tc cells were observed between the Cand G groupsThe DSS colitis groups had higher percentages

Table 2 Leukocyte populations in blood and mesenteric lymphnodes (MLNs) ()

C G DC DGBlood

Neutrophils 87 plusmn 05 80 plusmn 09 161 plusmn 27lowast 169 plusmn 21lowast

Monocytes 72 plusmn 09 67 plusmn 07 82 plusmn 13 77 plusmn 15T cells 119 plusmn 04 117 plusmn 04 93 plusmn 12 93 plusmn 14

B cells 503 plusmn 09 534 plusmn 31 532 plusmn 27 490 plusmn 21

Th cells 67 plusmn 16 64 plusmn 04 73 plusmn 12 81 plusmn 08

Tc cells 36 plusmn 08 44 plusmn 03 45 plusmn 02 41 plusmn 03

MLNsT cells 452 plusmn 24 467 plusmn 22 321 plusmn 23

lowast

349 plusmn 10lowast

B cells 303 plusmn 18 290 plusmn 33 313 plusmn 21 297 plusmn 14

Th cells 118 plusmn 03 123 plusmn 07 112 plusmn 09 121 plusmn 09

Tc cells 63 plusmn 04 67 plusmn 06 58 plusmn 03 65 plusmn 05

CD45-positive cells were considered to be leukocytes and gated to determinethe population of leukocytes using a flow cytometer Staining for Ly-6G F480 CD3120576 and CD19 was used to respectively identify neutrophilmonocyte T cell and B cell populations For the analysis of T cell subpop-ulations lymphocytes were gated on the basis of their forward-and side-scatter profiles Percentages of T helper (Th) and cytotoxic T (Tc) cells wererespectively determined by CD4-and CD8-expressing cells in lymphocytesValues are presented as the mean plusmn SEM lowastSignificantly differs from the Cgroup (119875 lt 005)

of PSGL-1- CD11a- and CCR9-expressing blood and MLNTc cells whereas expression levels of 12057241205737 integrins did notdiffer among the 4 groups Compared to the DC group theDG group had lower expression of CD11a by blood (Figures6(a)ndash6(d)) and MLN Tc cells (Figures 7(a)ndash7(d)) There wasno difference in expression levels of CCR9 on 12057241205737-positiveTc cells between the 2 DSS colitis groups (Figures 6(e) and7(e))

6 Mediators of Inflammation

0

10

20

30

40

50

C G DC DG

PSG

L-1

()

lowast

lowast

dagger

(a)

0

2

4

6

8

10

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

5

10

15

20

25

C G DC DG

dagger

lowast

CD11

a (

)

(c)

0

10

20

30

40

50

C G DC DG

dagger

lowast

lowastCCR9

(

)

(d)

0

15

30

45

60

C G DC DG

lowast

CCR

9 ex

pres

sing

dagger

CD4+12057241205737+

T ce

lls (

)

(e)

Figure 4 Percentage of adhesionmolecule- and chemokine receptor-expressingThelper (Th) cells in blood CD4-positive blood lymphocyteswere gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d)) (e) Expression of CCR9 by CD4and 12057241205737 integrin double-positive blood lymphocytes Values are shown as the mean plusmn SEM lowastSignificantly different from the C group(119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

35 Gene Expression of Endothelial Adhesion Molecules inColon Tissues There was no difference in mRNA levels ofICAM-1 P-selectin and E-selectin between the C and Ggroups in colon tissues DSS-induced colitis greatly upreg-ulated the adhesion molecule genes expressed by activatedendothelial cells Compared to the DC group the expression

levels of ICAM-1 P-selectin and E-selectin mRNA weresuppressed in the DG group (Figure 8)

36 T Lymphocyte Subsets in the Colon Mucosa CD3 is thecell surface marker of T cells CD3 and CD4 double-positivecells are considered Th cells whereas Tc cells coexpress CD3

Mediators of Inflammation 7

0

10

20

30

40

50

C G DC DG

PSG

L-1

()

lowast

lowast

dagger

(a)

0

2

4

6

8

10

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

10

20

30

40

50

C G DC DG

CD11

a (

)

dagger

lowast

(c)

0

10

20

30

40

50

C G DC DG

dagger

lowast

CCR9

(

)

(d)

0

15

30

45

60

C G DC DG

dagger

lowast

CCR

9 ex

pres

sing

CD4+12057241205737+

T ce

lls (

)

(e)

Figure 5 Percentage of adhesion molecule- and chemokine receptor-expressing T helper (Th) cells in mesenteric lymph nodes (MLNs)CD4-positiveMLN lymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d))(e) Expression of CCR9 by CD4 and 12057241205737 integrin double-positive MLN lymphocytes Values are shown as the mean plusmn SEM lowastSignificantlydifferent from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

andCD8As shown in Figure 9 the immunoreactive intensityof Th cells was higher in the DC group than the DG groupHowever intensities of Tc cell populations did not differbetween the DC and DG groups (Figure 10)

4 Discussion

DSS is a heparin-like polysaccharide which results in acutechemical toxicity that disrupts the intestinal epithelial cell

8 Mediators of Inflammation

0

10

20

30

40

50

C G DC DG

lowast lowast

PSG

L-1

()

(a)

0

5

10

15

20

25

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

5

10

15

20

25

C G DC DG

dagger

lowast

CD11

a (

)

(c)

0

10

20

30

40

50

C G DC DG

CCR9

()

lowast

lowast

(d)

0

20

40

60

80

100

C G DC DG

lowastlowast

CCR

9 ex

pres

sing

CD8+12057241205737+

T ce

lls (

)

(e)

Figure 6 Percentage of adhesion molecule- and chemokine receptor-expressing cytotoxic T (Tc) cells in blood CD8-positive bloodlymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d)) (e) Expressionof CCR9 by CD8 and 12057241205737 integrin double-positive blood lymphocytes Values are shown as the mean plusmn SEM lowastSignificantly different fromthe C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

barrier [21] DSS-induced colitis is characterized by extensivecrypt and epithelial cell damage with ulceration tissueedema and infiltration of immune cells predominantly in thedistal colon that mimics the histological features of UC [22]Recent studies indicated that DSS-induced morphological

and biochemical damage also extends to the small intestines[23] Susceptibilities to DSS-induced colitis differ in variousinbred mouse strains [24] A single cycle of DSS exposureto the C57BL6 strain was found to develop acute colitiswhich later proceeds to chronic inflammation and T cell

Mediators of Inflammation 9

C G DC DG

PSG

L-1

()

0

10

20

30

40

50

lowastlowast

(a)

CD11

a (

)

C G DC DG0

5

10

15

20

25

CD11

a (

)

12057241205737

inte

grin

s (

)

(b)

C G DC DG0

5

10

15

20

25

CD11

a (

)

dagger

lowast

(c)

CCR9

()

C G DC DG0

5

10

15

20

25

lowast

lowast

(d)

CCR

9 ex

pres

sing

0

15

30

45

60

lowastlowast

CD+ 812057241205737+

T ce

lls (

)

C G DC DG

(e)

Figure 7 Percentage of adhesion molecule- and chemokine receptor-expressing cytotoxic T (Tc) cells in mesenteric lymph nodes (MLNs)CD8-positiveMLN lymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d))(e) Expression of CCR9 by CD8 and 12057241205737 integrin double-positive MLN lymphocytes Values are shown as the mean plusmn SEM lowastSignificantlydifferent from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

migration to the colon plays an important role in theprogression to chronicity [25] Regarding the high sensitivityto DSS C57BL6 mice were used in this study to analyze theconsequences of DSS exposure on adhesion molecules and

chemokine receptors involved in T cell trafficking to theintestines

IBD is associated with a massive influx of immune cellsinto the gut Previous studies indicated that increased local

10 Mediators of Inflammation

ICAM-1 P-selectin E-selectin

Relat

ive m

RNA

ratio

0

50

100

150

200

1000

1250

1500

CG

DCDG

daggerdagger

dagger

lowast

lowastlowast

lowast

lowast

lowast

Figure 8 Gene expression of endothelial adhesion molecules incolon tissues mRNA levels were analyzed by a real-time PCR TheC group was used as a calibrator and the data were presented as thefold change in gene expression relative to the calibrator Values areshown as themean plusmn SEM lowastSignificantly different from the C group(119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

secretion of proinflammatory cytokines by the inflamedcolon leads to upregulated expressions of vascular adhesionmolecules resulting in a sustained influx of inflammatorycells [26] Although inflammation predominantly occurs inthe GI tract IBD patients are likely to develop extraintestinalmanifestations [27] that may be attributed to aberrant acti-vation and homing of T cells [28] A recent study indicatedthat circulating CD4+ and CD8+ T cells are activated in bothCD and UC patients and these cells were correlated withleakage of microbial products from the impaired intestinalbarrier [5] Also the severity of DSS-induced colitis wascorrelatedwith the immune response ofMLNs [29] A similarphenomenon was also observed in this study Our resultsindicated that expressions of adhesion molecules and CCR9on Th cells and Tc cells significantly increased in both bloodand MLNs after DSS exposure These findings suggest thatboth local and systemic T cells are activated The role of Thcells in the pathogenesis of IBD has been widely studiedDysregulation ofTh cells can lead to immune cell infiltrationinto the intestinal mucosa and cause persistent inflammation[3 6] The pathogenic role of Tc cells in IBD has been lessinvestigated A previous study showed that antigen-specificTc cells caused relapsing colitis in normal mice due to thecytolytic function against the intestinal epithelium [30] Leeet al [4] reported a gene expression profile of circulatingCD8+ T cells that predicted a more aggressive disease coursefor IBD patients However the modulatory mechanism of Tccells in IBD is still under investigation

Naıve T cells constantly recirculate between the bloodand secondary lymphoid organs Once activated in secondarylymphoid organs they become effector T cells that expressadhesion molecules and chemokine receptors which controltheir extravasation into nonlymphoid tissue sites [31] T cell

trafficking to the gut and gut-associated lymphoid tissues(GALTs) requires 12057241205737 integrins The ligand MAdCAM-1is constitutively expressed by the mucosal endothelium inthe small intestine and colon [32] CCR9 is thought toparticipate in the specific localization of T cells to the smallintestines because the sole ligand for CCR9 C-C chemokineligand 25 (CCL25) is strongly expressed by the small intesti-nal epithelium [33] However Wurbel et al [34] revealedthat CCL25 expression increased during the recovery phaseafter acute DSS administration suggesting a regulatory roleof CCR9CCL25 interactions during colonic inflammationIn this study percentages of PSGL-1- LFA-1- and CCR9-expressing T cells were upregulated in acute DSS colitiswhereas expression levels of 12057241205737 integrins in colitic micedid not differ from those of normal mice It is possible thatT cell trafficking into the gut during acute DSS exposure isless dependent on 12057241205737 integrins In support of our findingsWang et al [35] reported that localization of T cells to theintestines was relatively unaffected by 12057241205737 blockade duringacute DSS-induced colitis

Gln is a critical fuel source for enterocytes and immunecells Gln supplementation attenuates gut injury by a com-plex mechanism which involves protecting the epithelialbarrier function reducing oxidative stress and modulat-ing inflammatory responses [36] The local and systemicimmunomodulatory effects of Gln have been discussed invarious experimental colitis models via different adminis-tration routes Studies using rodents with trinitrobenzenesulfonic acid-induced colitis indicated that Gln given bythe rectal route inhibits nuclear factor (NF)-120581B- and STAT-mediated inflammation in colon tissues [15] and further pre-vents colon fibrosis through downregulating gene pathwaysthat contribute to the accumulation of matrix proteins [37]We recently demonstrated that intraperitoneal pretreatmentwith alanyl-Gln a Gln-containing dipeptide widely used inparenteral nutrition suppresses cytokine expression in bloodTh cells reduces NF-120581B-mediated inflammatory responsesin the colon and upregulates expressions of genes whichpromote recovery of the colonic mucosa [17 38]

Because most exogenous Gln is absorbed in the proximalsmall intestine it might not reach the inflamed colon at asufficient concentration tomodulate inflammatory responses[36] However the enteral route of Gln administrationstill showed protective effects against DSS-induced damageOral Gln supplementation reduced the feces water contentenhanced expression of HSPs in the colonic mucosa andameliorated colon injury caused byDSS exposure [16 39 40]A previous study indicated that oral Gln attenuated leukocyteadhesion and emigration in a rodentmodel of indomethacin-induced ileitis [41] In this study we demonstrated thatoral Gln administration suppressed adhesion molecules andCCR9 expressed by T cells and downregulated the mRNAlevels of adhesion molecules expressed by endothelium incolon tissues The histological findings also support theresults that Gln administration suppressed the infiltrationof Th cells into the colon mucosa Gln consumption is animportant component of T cell activation [42 43] Differentsusceptibilities ofThandTc cells toGln supplementationmaybe explained by a higher cell population of Th cells which

Mediators of Inflammation 11

50120583m

50120583m

50120583m

50120583m

CD3

C

G

DC

DG

(a)

50120583m

50120583m

50120583m

50120583m

CD4

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 9 Representative examples of the immunofluorescence staining of T helper cells In the left column cell surface antigens were stainedfor CD3 (green FITC) and staining of CD4 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI(blue) and the last column presents a colocalized fluorescence image of CD3+CD4+ T cells

competes as a Gln source with Tc cells Further studies areneeded to investigate the molecular mechanisms involved ingene expressions of adhesion molecules and CCR9 regulatedby Gln

In conclusion this study showed for the first time thatpretreatment with oral Gln reduced adhesion molecule- andCCR9-expressing T cells induced by DSS exposure Theinhibitory abilities against adhesion molecule and CCR9expressions were more obvious inTh cells than Tc cells AlsoGln supplementation reduced gene expressions of endothe-lial adhesion molecules in colons prevented BW loss and

attenuated colon edema in coliticmice Our results imply thatdietary Gln prevented Th cell trafficking into colon tissuesand provide a new mechanism of Gln supplementation thathas beneficial effects on ameliorating the severity of acuteDSS-induced colitis

Conflict of Interests

The authors declare that they have no conflict of interests

12 Mediators of Inflammation

50120583m

CD3

50120583m

50120583m

50120583m

C

G

DC

DG

(a)

CD8

50120583m

50120583m

50120583m

50120583m

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 10 Representative immunofluorescence images of cytotoxic T cells In the left column cell surface antigens were stained for CD3(green FITC) and the staining of CD8 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI (blue)and the last column presents a colocalized fluorescence image of CD3+CD8+ T cells

Acknowledgments

The authors would like to thank Man-Hui Pai Assistant Pro-fessor of Department of Anatomy Taipei Medical Universityfor her technical assistance in immunofluorescence stainingThis studywas supported by aResearchGrant (NSC100-2320-B-038-007-MY3) from the National Science Council TaipeiTaiwan

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] C T Murphy K Nally F Shanahan and S Melgar ldquoShining alight on intestinal trafficrdquoClinical and Developmental Immunol-ogy vol 2012 Article ID 808157 14 pages 2012

Mediators of Inflammation 13

[3] L A Zenewicz A Antov and R A Flavell ldquoCD4 T-cell differ-entiation and inflammatory bowel diseaserdquo Trends inMolecularMedicine vol 15 no 5 pp 199ndash207 2009

[4] J C Lee P A Lyons E F McKinney et al ldquoGene expressionprofiling of CD8+ T cells predicts prognosis in patients withCrohn disease and ulcerative colitisrdquo Journal of Clinical Inves-tigation vol 121 no 10 pp 4170ndash4179 2011

[5] N T Funderburg S R Stubblefield Park H C Sung et alldquoCirculating CD4+ and CD8+ T cells are activated in IBDand are associated with plasma markers of inflammationrdquoImmunology vol 140 no 1 pp 87ndash97 2013

[6] E J Villablanca B Cassani U H von Andrian and J RMora ldquoBlocking lymphocyte localization to the gastrointestinalmucosa as a therapeutic strategy for inflammatory boweldiseasesrdquo Gastroenterology vol 140 no 6 pp 1776ndash1784 2011

[7] R P McEver and R D Cummings ldquoPerspectives series celladhesion in vascular biology Role of PSGL-1 binding toselectins in leukocyte recruitmentrdquo The Journal of ClinicalInvestigation vol 100 no 3 pp 485ndash491 1997

[8] C Berlin E L Berg M J Briskin et al ldquo12057241205737 Integrin medi-ates lymphocyte binding to the mucosal vascular addressinMAdCAM-1rdquo Cell vol 74 no 1 pp 185ndash195 1993

[9] K Ley C Laudanna M I Cybulsky and S Nourshargh ldquoGet-ting to the site of inflammation the leukocyte adhesion cascadeupdatedrdquoNature Reviews Immunology vol 7 no 9 pp 678ndash6892007

[10] B Moser M Wolf A Walz and P Loetscher ldquoChemokinesmultiple levels of leukocyte migration controlrdquo Trends inImmunology vol 25 no 2 pp 75ndash84 2004

[11] S Arimilli W Ferlin N Solvason S Deshpande M Howardand S Mocci ldquoChemokines in autoimmune diseasesrdquo Immuno-logical Reviews vol 177 pp 43ndash51 2000

[12] C Koenecke and R Forster ldquoCCR9 and inflammatory boweldiseaserdquo Expert Opinion on Therapeutic Targets vol 13 no 3pp 297ndash306 2009

[13] B Johansson-Lindbom and W W Agace ldquoGeneration of gut-homing T cells and their localization to the small intestinalmucosardquo Immunological Reviews vol 215 no 1 pp 226ndash2422007

[14] P EWischmeyer ldquoGlutaminemode of action in critical illnessrdquoCritical Care Medicine vol 35 no 9 pp S541ndashS544 2007

[15] N A Kretzmann H Fillmann J L Mauriz C A MarroniJ Gonzalez-Gallego and M J Tunon ldquoEffects of glutamineon proinflammatory gene expression and activation of nuclearfactor Kappa B and signal transducers and activators oftranscription in TNBS-induced colitisrdquo Inflammatory BowelDiseases vol 14 no 11 pp 1504ndash1513 2008

[16] H Xue A J D Sufit and P EWischmeyer ldquoGlutamine therapyimproves outcome of in vitro and in vivo experimental colitismodelsrdquo Journal of Parenteral and Enteral Nutrition vol 35 no2 pp 188ndash197 2011

[17] C-C Chu Y-C Hou M-H Pai C-J Chao and S-L YehldquoPretreatment with alanyl-glutamine suppresses T-helper-cell-associated cytokine expression and reduces inflammatoryresponses in mice with acute DSS-induced colitisrdquo The Journalof Nutritional Biochemistry vol 23 no 9 pp 1092ndash1099 2011

[18] S Kew S M Wells P Yaqoob F A Wallace E A Miles and PC Calder ldquoDietary glutamine enhances murine T-lymphocyteresponsivenessrdquo The Journal of Nutrition vol 129 no 8 pp1524ndash1531 1999

[19] S M Wells S Kew P Yaqoob F A Wallace and P C CalderldquoDietary glutamine enhances cytokine production by murinemacrophagesrdquo Nutrition vol 15 no 11-12 pp 881ndash884 1999

[20] Y-C Hou M-H Pai W-C Chiu Y-M Hu and S-L YehldquoEffects of dietary glutamine supplementation on lung injuryinduced by lipopolysaccharide administrationrdquo American Jour-nal of PhysiologymdashLung Cellular and Molecular Physiology vol296 no 3 pp L288ndashL295 2009

[21] M Kawada A Arihiro and E Mizoguchi ldquoInsights fromadvances in research of chemically induced experimental mod-els of human inflammatory bowel diseaserdquo World Journal ofGastroenterology vol 13 no 42 pp 5581ndash5593 2007

[22] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[23] R Yazbeck G S Howarth R N Butler M S Geier and CA Abbott ldquoBiochemical and histological changes in the smallintestine of mice with dextran sulfate sodium colitisrdquo Journal ofCellular Physiology vol 226 no 12 pp 3219ndash3224 2011

[24] M Mahler I J Bristol E H Leiter et al ldquoDifferentialsusceptibility of inbred mouse strains to dextran sulfatesodium-induced colitisrdquo American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 274 no 3 part 1 ppG544ndashG551 1998

[25] S Melgar A Karlsson and E Michaelsson ldquoAcute colitisinduced by dextran sulfate sodium progresses to chronicityin C57BL6 but not in BALBc mice correlation betweensymptoms and inflammationrdquoAmerican Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 288 no 6 pp G1328ndashG1338 2005

[26] MKoizumiN King R Lobb C Benjamin andDK PodolskyldquoExpression of vascular adhesion molecules in inflammatorybowel diseaserdquo Gastroenterology vol 103 no 3 pp 840ndash8471992

[27] L Peyrin-Biroulet E V Loftus Jr J-F Colombel and W JSandborn ldquoLong-term complications extraintestinal manifes-tations and mortality in adult Crohnrsquos disease in population-based cohortsrdquo Inflammatory Bowel Diseases vol 17 no 1 pp471ndash478 2011

[28] D H Adams and B Eksteen ldquoAberrant homing of mucosal Tcells and extra-intestinal manifestations of inflammatory boweldiseaserdquoNature Reviews Immunology vol 6 no 3 pp 244ndash2512006

[29] TW Spahn H Herbst P D Rennert et al ldquoInduction of colitisinmice deficient of Peyerrsquos patches andmesenteric lymph nodesis associated with increased disease severity and formation ofcolonic lymphoid patchesrdquo The American Journal of Pathologyvol 161 no 6 pp 2273ndash2282 2002

[30] S Nancey S Holvoet I Graber et al ldquoCD8+ Cytotoxic T CellsInduce Relapsing Colitis in Normal Micerdquo Gastroenterologyvol 131 no 2 pp 485ndash496 2006

[31] E C Butcher and L J Picker ldquoLymphocyte homing and home-ostasisrdquo Science vol 272 no 5258 pp 60ndash66 1996

[32] C Berlin R F Bargatze J J Campbell et al ldquo1205724 Integrinsmediate lymphocyte attachment and rolling under physiologicflowrdquo Cell vol 80 no 3 pp 413ndash422 1995

[33] K A Papadakis J Prehn V Nelson et al ldquoThe role of thymus-expressed chemokine and its receptor CCR9 on lymphocytesin the regional specialization of the mucosal immune systemrdquoJournal of Immunology vol 165 no 9 pp 5069ndash5076 2000

14 Mediators of Inflammation

[34] M-A Wurbel M G McIntire P Dwyer and E FiebigerldquoCCL25CCR9 interactions regulate large intestinal inflamma-tion in a murine model of acute colitisrdquo PLoS ONE vol 6 no 1Article ID e16442 2011

[35] CWang E K Hanly LWWheeler M Kaur K GMcDonaldand R D Newberry ldquoEffect of 12057241205737 blockade on intestinallymphocyte subsets and lymphoid tissue developmentrdquo Inflam-matory Bowel Diseases vol 16 no 10 pp 1751ndash1762 2010

[36] M Coeffier R Marion-Letellier and P Dechelotte ldquoPotentialfor amino acids supplementation during inflammatory boweldiseasesrdquo Inflammatory Bowel Diseases vol 16 no 3 pp 518ndash524 2010

[37] B San-Miguel I Crespo N A Kretzmann et al ldquoGlutamineprevents fibrosis development in rats with colitis induced by246-trinitrobenzene sulfonic acidrdquo The Journal of Nutritionvol 140 no 6 pp 1065ndash1071 2010

[38] Y C Hou C C Chu T L Ko et al ldquoEffects of alanyl-glutaminedipeptide on the expression of colon-inflammatory mediatorsduring the recovery phase of colitis induced by dextran sulfatesodiumrdquo European Journal of Nutrition vol 52 no 3 pp 1089ndash1098 2013

[39] Y Shiomi S Nishiumi M Ooi et al ldquoGCMS-based meta-bolomic study in mice with colitis induced by dextran sulfatesodiumrdquo Inflammatory Bowel Diseases vol 17 no 11 pp 2261ndash2274 2011

[40] M Vicario C Amat M Rivero M Moreto and C PelegrıldquoDietary glutamine affects mucosal functions in rats with mildDSS-induced colitisrdquoThe Journal of Nutrition vol 137 no 8 pp1931ndash1937 2007

[41] H Arndt F Kullmann F Reuszlig J Scholmerich and K-D Pal-itzsch ldquoGlutamine attenuates leukocyte-endothelial cell adhe-sion in indomethacin-induced intestinal inflammation in theratrdquo Journal of Parenteral and Enteral Nutrition vol 23 no 1pp 12ndash18 1999

[42] P Yaqoob and P C Calder ldquoGlutamine requirement of prolif-erating T lymphocytesrdquo Nutrition vol 13 no 7-8 pp 646ndash6511997

[43] E L Carr A Kelman G S Wu et al ldquoGlutamine uptake andmetabolism are coordinately regulated by ERKMAPK duringT lymphocyte activationrdquo Journal of Immunology vol 185 no2 pp 1037ndash1044 2010

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Behavioural Neurology

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Disease Markers

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OncologyJournal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 3

monocytesmacrophages FITC-conjugated anti-Ly6G (BDBiosciences San Jose CA USA) for neutrophils APC-con-jugated anti-CD3120576 (eBioscience) for T cells and Pacific blue-conjugated anti-CD19 (Biolegend) for B cells Antibodieswere used at the concentration recommended by manufac-turer After a 30min incubation at 4∘C in the dark red bloodcells were lysed and cells were suspended in staining bufferand then analyzed with a FACS Canto II flow cytometer (BDBiosciences) CD45-positive cells were gated and results arepresented as a percentage of specific CD-marker-expressingcells in blood leukocytes Representative flow cytometry plotsare shown in Figure 2(a)

24 Lymphocyte Populations inMLNs Cell suspensions fromMLNs were obtained by passing the tissues through anylon cell strainer with a 40 120583m pore size (BD Biosciences)in RPMI1640 medium (Biological Industries Kibbutz BeitHaemek Israel) After centrifugation at 300timesg for 10minpelletedMLN cells were suspended in 1mL of staining bufferOne hundred microliters of cell suspension was incubatedwith APC-conjugated anti-CD3120576 (eBioscience) and Pacificblue-conjugated anti-CD19 (Biolegend) for 30min at 4∘Cin the dark Stained cells were washed and resuspended instaining buffer to measure the lymphocyte population byflow cytometry Percentages of T and B lymphocytes weredetermined by CD3120576- and CD19-expressing cells in MLNcells Representative flow cytometry plots are shown inFigure 2(b)

25 Expressions of Adhesion Molecules and Chemokine Recep-tors by T Cells Whole blood and MLNs were used to ana-lyze adhesion molecule- and chemokine receptor-express-ing T cells Whole blood and MLN cells obtained asdescribed above were split into 2 vials with 100 120583L ineach aliquot and these were incubated with Pacific blue-conjugated anti-CD4 (BD Biosciences) or Pacific blue-conjugated anti-CD8 antibodies (Biolegend) To investigateexpressions of adhesion molecules and chemokine receptorsPE-conjugated anti-PSGL-1 antibodies (BD Biosciences)APC-conjugated anti-12057241205737 integrin FITC-conjugated anti-CD11a and PerCP-Cy55-conjugated anti-CCR9 (Biolegend)were added Stained cells were analyzed by five-color flowcytometry Lymphocytes were gated on the basis of theirforward- and side-scatter profiles Fluorescence data wererecorded and results are presented as percentages of adhesionmolecule- and chemokine receptor-expressing CD4+ andCD8+ lymphocytes Representative flow cytometry plots areshown in Figure 2(c)

26 RNA Extraction and Real-Time PCR Total RNA wasisolated from colon tissue using the Trizol reagent (Invi-trogen Carlsbad CA) RNA (1120583g) was reverse-transcribedwith a complementary (c)DNA synthesis kit (FermentasGlen Burnie MD USA) according to standard protocols Forreal-time PCR 5 120583L of 110 diluted cDNA was amplified in a25 120583L PCR volume containing 125 120583L of 2X SYBR greenmas-ter mix reagent (Applied Biosystems Foster City CA USA)The reaction was performed with ABI 7300 Real-Time PCRSystem (Applied Biosystems) according to the thermocycling

protocol recommended by the PCR system Primer sequenceswere as follows mouse ICAM-1 (51015840-AGCACCTCCCCA-CCTACTTT-31015840 and 51015840-AGCTTGCACGACCCTTCTAA-31015840) mouse P-selectin (51015840-TCCAGGAAGCTCTGACGT-ACTTG-31015840 and 51015840-GCAGCGTTAGTGAAGACTCCGTAT-31015840) mouse E-selectin (51015840-TGAACTGAAGGGATCAAG-AAGACT-31015840 and 51015840-GCCGAGGGACATCATCACAT-31015840)and mouse 18S rRNA (51015840-CGCGGTTCTATTTTGTTGGT31015840and 51015840-AGTCGGCATCGTTTATGGTC-31015840) All sampleswere analyzed in triplicate and fold change for each targetgene was calculated by the equation 2minusΔΔCt (ΔCt indicatesthe difference in threshold cycles between the test gene and18S rRNA and ΔΔCt indicates the difference of ΔCt betweenthe experimental and C groups)

27 Immunofluorescence Staining Double-staining combina-tions CD3-CD4 and CD3-CD8 were performed on 5 120583mparaffin-embedded colon sections After antigen retrievalsections were incubated with an antibody against CD3120576(Santa Cruz Biotechnology Santa Cruz CA USA) overnightat 4∘C and amplified with a rabbit anti-goat immunoglobulinG (IgG) secondary antibody conjugated with FITC (SantaCruz Biotechnology) For colocalization sections were thencostained overnight at 4∘Cwith secondary antibodies againstCD4 (Abcam Cambridge UK) or CD8 (Novus BiologicalsLittleton CO USA) and amplified with the respective appro-priate secondary antibodies goat anti-mouse IgG or goatanti-rabbit IgG conjugated with rhodamine (Santa CruzBiotechnology) Cell nuclei were counterstained with 410158406-diamidino-2-phenylindole (DAPI Sigma St Louis MOUSA) for 10min at room temperature Digital images at 400xmagnification per section were acquired using appropriatefilters of a Zeiss Axiophot fluorescence microscope (CarlZeiss MicroImaging LLC Thornwood NY USA) fitted witha Nikon D1X digital camera (Tokyo Japan) Cells containingboth FITC and rhodamine labels appeared yellow Theseimages were then overlaid with DAPI-staining images todetermine the infiltration of T lymphocyte subpopulations inthe colon mucosa

28 Statistical Analysis All data are expressed as the mean plusmnstandard error of themean (SEM) Differences among groupswere analyzed by an analysis of variance (ANOVA) withTukeyrsquos test A two-way ANOVA with Bonferroni correctionwas used to analyze differences in BW changes A 119875 value oflt005 was considered statistically significant

3 Results

31 BW and WeightLength Ratio of the Colon Initial BWsranged 21sim25 g and did not differ among the 4 groupsThere was no significant difference in BWs during the studybetween the C and G groups At 4 d (d 9) and 5 d (d 10) afterDSS administration weight loss was observed in the DCgroup compared to the C group whereas mice with Glnsupplementationmaintained their BWsduring theDSS expo-sure period At the end of the study BWs were significantlyhigher in the DG group than the DC group (Figure 3(a))

4 Mediators of Inflammation

81 80

F480

C

C

G

G

DC DG

DC DG

102

102

103

103

104

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

105

102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105

102 103 104 105 102 103 104 105 102 103 104 105

169 159

Ly6G Ly6G Ly6G Ly6G

CD19

CD3120576 CD3120576 CD3120576 CD3120576

Blood leukocytes

(a)

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105

C G DC DG

CD3120576 CD3120576 CD3120576 CD3120576

CD19

456 463 332 348

MLN cells

(b)

102 103 104 105

Gated cells

Gated cells

Gated cells

FSC

FSC

CD4 or CD8

PSGL-1 12057241205737

12057241205737

CD11a CCR9

CCR9

Cou

nt

Cou

nt

(c)

Figure 2 Representative flow cytometry plots Blood leukocytes (a) were defined by gating on CD45-positive cells The percentage of Ly6G-positive neutrophils from an individual representative mouse per group is listed For analyzing the lymphocyte population in MLNs (b)MLN cells were first gated to exclude debris Numbers indicate the percentage of CD3120576-positive lymphocytes in MLN cells For analyzingthe expression of adhesion molecules and chemokine receptors by T cells (c) lymphocytes were first identified based on low FSC and SSCcharacteristics CD4- or CD8-positive lymphocytes were gated to analyze the percentages of adhesion molecule- and chemokine receptor-expressing CD4+ and CD8+ lymphocytes Representative dot plots of leukocytes in blood are shown

Mediators of Inflammation 5

0 1 2 3 4 5 6 7 8 9 10

Body

wei

ght (

g)

0

18

20

22

24

26

28

30

CG

DC

Days

DG

daggerlowast

lowast

(a)

C G DC DG

Col

on w

eigh

tlen

gth

ratio

(gc

m)

000

002

004

006

008

010

daggerlowast

lowast

(b)

Figure 3 Body weight (a) and weightlength ratio of the colon (b) Data are presented as the mean plusmn SEM C normal mice fed the controldiet G normal mice fed a glutamine-enriched diet DC DSS group fed the control diet DG DSS group fed a glutamine-enriched dietlowastSignificantly different from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

The weightlength ratio of the colon an indicator of colonicedema was significantly higher in the colitis groups than theC group (Figure 3(b)) Treatment withGln attenuated colonicedema associated with DSS-induced inflammation

32 Leukocyte Populations in Blood and MLNs There wasno significant difference in blood or MLN leukocyte sub-populations between the C and G groups Compared to theC group the DSS colitis groups had a higher percentage ofblood neutrophils and lower T-cell population inMLNs DSSexposure did not alter the blood monocyte and lymphocytedistributions Also subsets of effector T cells in the blood andMLNs did not change Gln supplementation had no influenceon blood leukocyte or MLN lymphocyte populations innormal or colitic mice (Table 2)

33 Adhesion Molecule and CCR9 Expressions by Th CellsPercentages of adhesion molecules and CCR9 expressed byblood and MLN Th cells did not differ between the C and Ggroups DSS administration resulted in higher PSGL-1 CD11a(LFA-1 120572L subunit) and CCR9 expressions byThcells in bothblood and MLNs whereas no difference in 12057241205737 integrinexpression was detected among the control and DSS groupsMice in the DG group had lower percentages of PSGL-1-CD11a- and CCR9-expressing Th cells in blood (Figures4(a)ndash4(d)) and MLNs (Figures 5(a)ndash5(d)) The expressionlevel of CCR9 on 12057241205737-positive Th cells was also suppressedin the DG group (Figures 4(e) and 5(e))

34 Adhesion Molecule and CCR9 Expressions by Tc CellsNo differences in adhesion molecules and CCR9 expressedby blood and MLN Tc cells were observed between the Cand G groupsThe DSS colitis groups had higher percentages

Table 2 Leukocyte populations in blood and mesenteric lymphnodes (MLNs) ()

C G DC DGBlood

Neutrophils 87 plusmn 05 80 plusmn 09 161 plusmn 27lowast 169 plusmn 21lowast

Monocytes 72 plusmn 09 67 plusmn 07 82 plusmn 13 77 plusmn 15T cells 119 plusmn 04 117 plusmn 04 93 plusmn 12 93 plusmn 14

B cells 503 plusmn 09 534 plusmn 31 532 plusmn 27 490 plusmn 21

Th cells 67 plusmn 16 64 plusmn 04 73 plusmn 12 81 plusmn 08

Tc cells 36 plusmn 08 44 plusmn 03 45 plusmn 02 41 plusmn 03

MLNsT cells 452 plusmn 24 467 plusmn 22 321 plusmn 23

lowast

349 plusmn 10lowast

B cells 303 plusmn 18 290 plusmn 33 313 plusmn 21 297 plusmn 14

Th cells 118 plusmn 03 123 plusmn 07 112 plusmn 09 121 plusmn 09

Tc cells 63 plusmn 04 67 plusmn 06 58 plusmn 03 65 plusmn 05

CD45-positive cells were considered to be leukocytes and gated to determinethe population of leukocytes using a flow cytometer Staining for Ly-6G F480 CD3120576 and CD19 was used to respectively identify neutrophilmonocyte T cell and B cell populations For the analysis of T cell subpop-ulations lymphocytes were gated on the basis of their forward-and side-scatter profiles Percentages of T helper (Th) and cytotoxic T (Tc) cells wererespectively determined by CD4-and CD8-expressing cells in lymphocytesValues are presented as the mean plusmn SEM lowastSignificantly differs from the Cgroup (119875 lt 005)

of PSGL-1- CD11a- and CCR9-expressing blood and MLNTc cells whereas expression levels of 12057241205737 integrins did notdiffer among the 4 groups Compared to the DC group theDG group had lower expression of CD11a by blood (Figures6(a)ndash6(d)) and MLN Tc cells (Figures 7(a)ndash7(d)) There wasno difference in expression levels of CCR9 on 12057241205737-positiveTc cells between the 2 DSS colitis groups (Figures 6(e) and7(e))

6 Mediators of Inflammation

0

10

20

30

40

50

C G DC DG

PSG

L-1

()

lowast

lowast

dagger

(a)

0

2

4

6

8

10

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

5

10

15

20

25

C G DC DG

dagger

lowast

CD11

a (

)

(c)

0

10

20

30

40

50

C G DC DG

dagger

lowast

lowastCCR9

(

)

(d)

0

15

30

45

60

C G DC DG

lowast

CCR

9 ex

pres

sing

dagger

CD4+12057241205737+

T ce

lls (

)

(e)

Figure 4 Percentage of adhesionmolecule- and chemokine receptor-expressingThelper (Th) cells in blood CD4-positive blood lymphocyteswere gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d)) (e) Expression of CCR9 by CD4and 12057241205737 integrin double-positive blood lymphocytes Values are shown as the mean plusmn SEM lowastSignificantly different from the C group(119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

35 Gene Expression of Endothelial Adhesion Molecules inColon Tissues There was no difference in mRNA levels ofICAM-1 P-selectin and E-selectin between the C and Ggroups in colon tissues DSS-induced colitis greatly upreg-ulated the adhesion molecule genes expressed by activatedendothelial cells Compared to the DC group the expression

levels of ICAM-1 P-selectin and E-selectin mRNA weresuppressed in the DG group (Figure 8)

36 T Lymphocyte Subsets in the Colon Mucosa CD3 is thecell surface marker of T cells CD3 and CD4 double-positivecells are considered Th cells whereas Tc cells coexpress CD3

Mediators of Inflammation 7

0

10

20

30

40

50

C G DC DG

PSG

L-1

()

lowast

lowast

dagger

(a)

0

2

4

6

8

10

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

10

20

30

40

50

C G DC DG

CD11

a (

)

dagger

lowast

(c)

0

10

20

30

40

50

C G DC DG

dagger

lowast

CCR9

(

)

(d)

0

15

30

45

60

C G DC DG

dagger

lowast

CCR

9 ex

pres

sing

CD4+12057241205737+

T ce

lls (

)

(e)

Figure 5 Percentage of adhesion molecule- and chemokine receptor-expressing T helper (Th) cells in mesenteric lymph nodes (MLNs)CD4-positiveMLN lymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d))(e) Expression of CCR9 by CD4 and 12057241205737 integrin double-positive MLN lymphocytes Values are shown as the mean plusmn SEM lowastSignificantlydifferent from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

andCD8As shown in Figure 9 the immunoreactive intensityof Th cells was higher in the DC group than the DG groupHowever intensities of Tc cell populations did not differbetween the DC and DG groups (Figure 10)

4 Discussion

DSS is a heparin-like polysaccharide which results in acutechemical toxicity that disrupts the intestinal epithelial cell

8 Mediators of Inflammation

0

10

20

30

40

50

C G DC DG

lowast lowast

PSG

L-1

()

(a)

0

5

10

15

20

25

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

5

10

15

20

25

C G DC DG

dagger

lowast

CD11

a (

)

(c)

0

10

20

30

40

50

C G DC DG

CCR9

()

lowast

lowast

(d)

0

20

40

60

80

100

C G DC DG

lowastlowast

CCR

9 ex

pres

sing

CD8+12057241205737+

T ce

lls (

)

(e)

Figure 6 Percentage of adhesion molecule- and chemokine receptor-expressing cytotoxic T (Tc) cells in blood CD8-positive bloodlymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d)) (e) Expressionof CCR9 by CD8 and 12057241205737 integrin double-positive blood lymphocytes Values are shown as the mean plusmn SEM lowastSignificantly different fromthe C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

barrier [21] DSS-induced colitis is characterized by extensivecrypt and epithelial cell damage with ulceration tissueedema and infiltration of immune cells predominantly in thedistal colon that mimics the histological features of UC [22]Recent studies indicated that DSS-induced morphological

and biochemical damage also extends to the small intestines[23] Susceptibilities to DSS-induced colitis differ in variousinbred mouse strains [24] A single cycle of DSS exposureto the C57BL6 strain was found to develop acute colitiswhich later proceeds to chronic inflammation and T cell

Mediators of Inflammation 9

C G DC DG

PSG

L-1

()

0

10

20

30

40

50

lowastlowast

(a)

CD11

a (

)

C G DC DG0

5

10

15

20

25

CD11

a (

)

12057241205737

inte

grin

s (

)

(b)

C G DC DG0

5

10

15

20

25

CD11

a (

)

dagger

lowast

(c)

CCR9

()

C G DC DG0

5

10

15

20

25

lowast

lowast

(d)

CCR

9 ex

pres

sing

0

15

30

45

60

lowastlowast

CD+ 812057241205737+

T ce

lls (

)

C G DC DG

(e)

Figure 7 Percentage of adhesion molecule- and chemokine receptor-expressing cytotoxic T (Tc) cells in mesenteric lymph nodes (MLNs)CD8-positiveMLN lymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d))(e) Expression of CCR9 by CD8 and 12057241205737 integrin double-positive MLN lymphocytes Values are shown as the mean plusmn SEM lowastSignificantlydifferent from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

migration to the colon plays an important role in theprogression to chronicity [25] Regarding the high sensitivityto DSS C57BL6 mice were used in this study to analyze theconsequences of DSS exposure on adhesion molecules and

chemokine receptors involved in T cell trafficking to theintestines

IBD is associated with a massive influx of immune cellsinto the gut Previous studies indicated that increased local

10 Mediators of Inflammation

ICAM-1 P-selectin E-selectin

Relat

ive m

RNA

ratio

0

50

100

150

200

1000

1250

1500

CG

DCDG

daggerdagger

dagger

lowast

lowastlowast

lowast

lowast

lowast

Figure 8 Gene expression of endothelial adhesion molecules incolon tissues mRNA levels were analyzed by a real-time PCR TheC group was used as a calibrator and the data were presented as thefold change in gene expression relative to the calibrator Values areshown as themean plusmn SEM lowastSignificantly different from the C group(119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

secretion of proinflammatory cytokines by the inflamedcolon leads to upregulated expressions of vascular adhesionmolecules resulting in a sustained influx of inflammatorycells [26] Although inflammation predominantly occurs inthe GI tract IBD patients are likely to develop extraintestinalmanifestations [27] that may be attributed to aberrant acti-vation and homing of T cells [28] A recent study indicatedthat circulating CD4+ and CD8+ T cells are activated in bothCD and UC patients and these cells were correlated withleakage of microbial products from the impaired intestinalbarrier [5] Also the severity of DSS-induced colitis wascorrelatedwith the immune response ofMLNs [29] A similarphenomenon was also observed in this study Our resultsindicated that expressions of adhesion molecules and CCR9on Th cells and Tc cells significantly increased in both bloodand MLNs after DSS exposure These findings suggest thatboth local and systemic T cells are activated The role of Thcells in the pathogenesis of IBD has been widely studiedDysregulation ofTh cells can lead to immune cell infiltrationinto the intestinal mucosa and cause persistent inflammation[3 6] The pathogenic role of Tc cells in IBD has been lessinvestigated A previous study showed that antigen-specificTc cells caused relapsing colitis in normal mice due to thecytolytic function against the intestinal epithelium [30] Leeet al [4] reported a gene expression profile of circulatingCD8+ T cells that predicted a more aggressive disease coursefor IBD patients However the modulatory mechanism of Tccells in IBD is still under investigation

Naıve T cells constantly recirculate between the bloodand secondary lymphoid organs Once activated in secondarylymphoid organs they become effector T cells that expressadhesion molecules and chemokine receptors which controltheir extravasation into nonlymphoid tissue sites [31] T cell

trafficking to the gut and gut-associated lymphoid tissues(GALTs) requires 12057241205737 integrins The ligand MAdCAM-1is constitutively expressed by the mucosal endothelium inthe small intestine and colon [32] CCR9 is thought toparticipate in the specific localization of T cells to the smallintestines because the sole ligand for CCR9 C-C chemokineligand 25 (CCL25) is strongly expressed by the small intesti-nal epithelium [33] However Wurbel et al [34] revealedthat CCL25 expression increased during the recovery phaseafter acute DSS administration suggesting a regulatory roleof CCR9CCL25 interactions during colonic inflammationIn this study percentages of PSGL-1- LFA-1- and CCR9-expressing T cells were upregulated in acute DSS colitiswhereas expression levels of 12057241205737 integrins in colitic micedid not differ from those of normal mice It is possible thatT cell trafficking into the gut during acute DSS exposure isless dependent on 12057241205737 integrins In support of our findingsWang et al [35] reported that localization of T cells to theintestines was relatively unaffected by 12057241205737 blockade duringacute DSS-induced colitis

Gln is a critical fuel source for enterocytes and immunecells Gln supplementation attenuates gut injury by a com-plex mechanism which involves protecting the epithelialbarrier function reducing oxidative stress and modulat-ing inflammatory responses [36] The local and systemicimmunomodulatory effects of Gln have been discussed invarious experimental colitis models via different adminis-tration routes Studies using rodents with trinitrobenzenesulfonic acid-induced colitis indicated that Gln given bythe rectal route inhibits nuclear factor (NF)-120581B- and STAT-mediated inflammation in colon tissues [15] and further pre-vents colon fibrosis through downregulating gene pathwaysthat contribute to the accumulation of matrix proteins [37]We recently demonstrated that intraperitoneal pretreatmentwith alanyl-Gln a Gln-containing dipeptide widely used inparenteral nutrition suppresses cytokine expression in bloodTh cells reduces NF-120581B-mediated inflammatory responsesin the colon and upregulates expressions of genes whichpromote recovery of the colonic mucosa [17 38]

Because most exogenous Gln is absorbed in the proximalsmall intestine it might not reach the inflamed colon at asufficient concentration tomodulate inflammatory responses[36] However the enteral route of Gln administrationstill showed protective effects against DSS-induced damageOral Gln supplementation reduced the feces water contentenhanced expression of HSPs in the colonic mucosa andameliorated colon injury caused byDSS exposure [16 39 40]A previous study indicated that oral Gln attenuated leukocyteadhesion and emigration in a rodentmodel of indomethacin-induced ileitis [41] In this study we demonstrated thatoral Gln administration suppressed adhesion molecules andCCR9 expressed by T cells and downregulated the mRNAlevels of adhesion molecules expressed by endothelium incolon tissues The histological findings also support theresults that Gln administration suppressed the infiltrationof Th cells into the colon mucosa Gln consumption is animportant component of T cell activation [42 43] Differentsusceptibilities ofThandTc cells toGln supplementationmaybe explained by a higher cell population of Th cells which

Mediators of Inflammation 11

50120583m

50120583m

50120583m

50120583m

CD3

C

G

DC

DG

(a)

50120583m

50120583m

50120583m

50120583m

CD4

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 9 Representative examples of the immunofluorescence staining of T helper cells In the left column cell surface antigens were stainedfor CD3 (green FITC) and staining of CD4 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI(blue) and the last column presents a colocalized fluorescence image of CD3+CD4+ T cells

competes as a Gln source with Tc cells Further studies areneeded to investigate the molecular mechanisms involved ingene expressions of adhesion molecules and CCR9 regulatedby Gln

In conclusion this study showed for the first time thatpretreatment with oral Gln reduced adhesion molecule- andCCR9-expressing T cells induced by DSS exposure Theinhibitory abilities against adhesion molecule and CCR9expressions were more obvious inTh cells than Tc cells AlsoGln supplementation reduced gene expressions of endothe-lial adhesion molecules in colons prevented BW loss and

attenuated colon edema in coliticmice Our results imply thatdietary Gln prevented Th cell trafficking into colon tissuesand provide a new mechanism of Gln supplementation thathas beneficial effects on ameliorating the severity of acuteDSS-induced colitis

Conflict of Interests

The authors declare that they have no conflict of interests

12 Mediators of Inflammation

50120583m

CD3

50120583m

50120583m

50120583m

C

G

DC

DG

(a)

CD8

50120583m

50120583m

50120583m

50120583m

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 10 Representative immunofluorescence images of cytotoxic T cells In the left column cell surface antigens were stained for CD3(green FITC) and the staining of CD8 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI (blue)and the last column presents a colocalized fluorescence image of CD3+CD8+ T cells

Acknowledgments

The authors would like to thank Man-Hui Pai Assistant Pro-fessor of Department of Anatomy Taipei Medical Universityfor her technical assistance in immunofluorescence stainingThis studywas supported by aResearchGrant (NSC100-2320-B-038-007-MY3) from the National Science Council TaipeiTaiwan

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] C T Murphy K Nally F Shanahan and S Melgar ldquoShining alight on intestinal trafficrdquoClinical and Developmental Immunol-ogy vol 2012 Article ID 808157 14 pages 2012

Mediators of Inflammation 13

[3] L A Zenewicz A Antov and R A Flavell ldquoCD4 T-cell differ-entiation and inflammatory bowel diseaserdquo Trends inMolecularMedicine vol 15 no 5 pp 199ndash207 2009

[4] J C Lee P A Lyons E F McKinney et al ldquoGene expressionprofiling of CD8+ T cells predicts prognosis in patients withCrohn disease and ulcerative colitisrdquo Journal of Clinical Inves-tigation vol 121 no 10 pp 4170ndash4179 2011

[5] N T Funderburg S R Stubblefield Park H C Sung et alldquoCirculating CD4+ and CD8+ T cells are activated in IBDand are associated with plasma markers of inflammationrdquoImmunology vol 140 no 1 pp 87ndash97 2013

[6] E J Villablanca B Cassani U H von Andrian and J RMora ldquoBlocking lymphocyte localization to the gastrointestinalmucosa as a therapeutic strategy for inflammatory boweldiseasesrdquo Gastroenterology vol 140 no 6 pp 1776ndash1784 2011

[7] R P McEver and R D Cummings ldquoPerspectives series celladhesion in vascular biology Role of PSGL-1 binding toselectins in leukocyte recruitmentrdquo The Journal of ClinicalInvestigation vol 100 no 3 pp 485ndash491 1997

[8] C Berlin E L Berg M J Briskin et al ldquo12057241205737 Integrin medi-ates lymphocyte binding to the mucosal vascular addressinMAdCAM-1rdquo Cell vol 74 no 1 pp 185ndash195 1993

[9] K Ley C Laudanna M I Cybulsky and S Nourshargh ldquoGet-ting to the site of inflammation the leukocyte adhesion cascadeupdatedrdquoNature Reviews Immunology vol 7 no 9 pp 678ndash6892007

[10] B Moser M Wolf A Walz and P Loetscher ldquoChemokinesmultiple levels of leukocyte migration controlrdquo Trends inImmunology vol 25 no 2 pp 75ndash84 2004

[11] S Arimilli W Ferlin N Solvason S Deshpande M Howardand S Mocci ldquoChemokines in autoimmune diseasesrdquo Immuno-logical Reviews vol 177 pp 43ndash51 2000

[12] C Koenecke and R Forster ldquoCCR9 and inflammatory boweldiseaserdquo Expert Opinion on Therapeutic Targets vol 13 no 3pp 297ndash306 2009

[13] B Johansson-Lindbom and W W Agace ldquoGeneration of gut-homing T cells and their localization to the small intestinalmucosardquo Immunological Reviews vol 215 no 1 pp 226ndash2422007

[14] P EWischmeyer ldquoGlutaminemode of action in critical illnessrdquoCritical Care Medicine vol 35 no 9 pp S541ndashS544 2007

[15] N A Kretzmann H Fillmann J L Mauriz C A MarroniJ Gonzalez-Gallego and M J Tunon ldquoEffects of glutamineon proinflammatory gene expression and activation of nuclearfactor Kappa B and signal transducers and activators oftranscription in TNBS-induced colitisrdquo Inflammatory BowelDiseases vol 14 no 11 pp 1504ndash1513 2008

[16] H Xue A J D Sufit and P EWischmeyer ldquoGlutamine therapyimproves outcome of in vitro and in vivo experimental colitismodelsrdquo Journal of Parenteral and Enteral Nutrition vol 35 no2 pp 188ndash197 2011

[17] C-C Chu Y-C Hou M-H Pai C-J Chao and S-L YehldquoPretreatment with alanyl-glutamine suppresses T-helper-cell-associated cytokine expression and reduces inflammatoryresponses in mice with acute DSS-induced colitisrdquo The Journalof Nutritional Biochemistry vol 23 no 9 pp 1092ndash1099 2011

[18] S Kew S M Wells P Yaqoob F A Wallace E A Miles and PC Calder ldquoDietary glutamine enhances murine T-lymphocyteresponsivenessrdquo The Journal of Nutrition vol 129 no 8 pp1524ndash1531 1999

[19] S M Wells S Kew P Yaqoob F A Wallace and P C CalderldquoDietary glutamine enhances cytokine production by murinemacrophagesrdquo Nutrition vol 15 no 11-12 pp 881ndash884 1999

[20] Y-C Hou M-H Pai W-C Chiu Y-M Hu and S-L YehldquoEffects of dietary glutamine supplementation on lung injuryinduced by lipopolysaccharide administrationrdquo American Jour-nal of PhysiologymdashLung Cellular and Molecular Physiology vol296 no 3 pp L288ndashL295 2009

[21] M Kawada A Arihiro and E Mizoguchi ldquoInsights fromadvances in research of chemically induced experimental mod-els of human inflammatory bowel diseaserdquo World Journal ofGastroenterology vol 13 no 42 pp 5581ndash5593 2007

[22] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[23] R Yazbeck G S Howarth R N Butler M S Geier and CA Abbott ldquoBiochemical and histological changes in the smallintestine of mice with dextran sulfate sodium colitisrdquo Journal ofCellular Physiology vol 226 no 12 pp 3219ndash3224 2011

[24] M Mahler I J Bristol E H Leiter et al ldquoDifferentialsusceptibility of inbred mouse strains to dextran sulfatesodium-induced colitisrdquo American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 274 no 3 part 1 ppG544ndashG551 1998

[25] S Melgar A Karlsson and E Michaelsson ldquoAcute colitisinduced by dextran sulfate sodium progresses to chronicityin C57BL6 but not in BALBc mice correlation betweensymptoms and inflammationrdquoAmerican Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 288 no 6 pp G1328ndashG1338 2005

[26] MKoizumiN King R Lobb C Benjamin andDK PodolskyldquoExpression of vascular adhesion molecules in inflammatorybowel diseaserdquo Gastroenterology vol 103 no 3 pp 840ndash8471992

[27] L Peyrin-Biroulet E V Loftus Jr J-F Colombel and W JSandborn ldquoLong-term complications extraintestinal manifes-tations and mortality in adult Crohnrsquos disease in population-based cohortsrdquo Inflammatory Bowel Diseases vol 17 no 1 pp471ndash478 2011

[28] D H Adams and B Eksteen ldquoAberrant homing of mucosal Tcells and extra-intestinal manifestations of inflammatory boweldiseaserdquoNature Reviews Immunology vol 6 no 3 pp 244ndash2512006

[29] TW Spahn H Herbst P D Rennert et al ldquoInduction of colitisinmice deficient of Peyerrsquos patches andmesenteric lymph nodesis associated with increased disease severity and formation ofcolonic lymphoid patchesrdquo The American Journal of Pathologyvol 161 no 6 pp 2273ndash2282 2002

[30] S Nancey S Holvoet I Graber et al ldquoCD8+ Cytotoxic T CellsInduce Relapsing Colitis in Normal Micerdquo Gastroenterologyvol 131 no 2 pp 485ndash496 2006

[31] E C Butcher and L J Picker ldquoLymphocyte homing and home-ostasisrdquo Science vol 272 no 5258 pp 60ndash66 1996

[32] C Berlin R F Bargatze J J Campbell et al ldquo1205724 Integrinsmediate lymphocyte attachment and rolling under physiologicflowrdquo Cell vol 80 no 3 pp 413ndash422 1995

[33] K A Papadakis J Prehn V Nelson et al ldquoThe role of thymus-expressed chemokine and its receptor CCR9 on lymphocytesin the regional specialization of the mucosal immune systemrdquoJournal of Immunology vol 165 no 9 pp 5069ndash5076 2000

14 Mediators of Inflammation

[34] M-A Wurbel M G McIntire P Dwyer and E FiebigerldquoCCL25CCR9 interactions regulate large intestinal inflamma-tion in a murine model of acute colitisrdquo PLoS ONE vol 6 no 1Article ID e16442 2011

[35] CWang E K Hanly LWWheeler M Kaur K GMcDonaldand R D Newberry ldquoEffect of 12057241205737 blockade on intestinallymphocyte subsets and lymphoid tissue developmentrdquo Inflam-matory Bowel Diseases vol 16 no 10 pp 1751ndash1762 2010

[36] M Coeffier R Marion-Letellier and P Dechelotte ldquoPotentialfor amino acids supplementation during inflammatory boweldiseasesrdquo Inflammatory Bowel Diseases vol 16 no 3 pp 518ndash524 2010

[37] B San-Miguel I Crespo N A Kretzmann et al ldquoGlutamineprevents fibrosis development in rats with colitis induced by246-trinitrobenzene sulfonic acidrdquo The Journal of Nutritionvol 140 no 6 pp 1065ndash1071 2010

[38] Y C Hou C C Chu T L Ko et al ldquoEffects of alanyl-glutaminedipeptide on the expression of colon-inflammatory mediatorsduring the recovery phase of colitis induced by dextran sulfatesodiumrdquo European Journal of Nutrition vol 52 no 3 pp 1089ndash1098 2013

[39] Y Shiomi S Nishiumi M Ooi et al ldquoGCMS-based meta-bolomic study in mice with colitis induced by dextran sulfatesodiumrdquo Inflammatory Bowel Diseases vol 17 no 11 pp 2261ndash2274 2011

[40] M Vicario C Amat M Rivero M Moreto and C PelegrıldquoDietary glutamine affects mucosal functions in rats with mildDSS-induced colitisrdquoThe Journal of Nutrition vol 137 no 8 pp1931ndash1937 2007

[41] H Arndt F Kullmann F Reuszlig J Scholmerich and K-D Pal-itzsch ldquoGlutamine attenuates leukocyte-endothelial cell adhe-sion in indomethacin-induced intestinal inflammation in theratrdquo Journal of Parenteral and Enteral Nutrition vol 23 no 1pp 12ndash18 1999

[42] P Yaqoob and P C Calder ldquoGlutamine requirement of prolif-erating T lymphocytesrdquo Nutrition vol 13 no 7-8 pp 646ndash6511997

[43] E L Carr A Kelman G S Wu et al ldquoGlutamine uptake andmetabolism are coordinately regulated by ERKMAPK duringT lymphocyte activationrdquo Journal of Immunology vol 185 no2 pp 1037ndash1044 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Mediators of Inflammation

81 80

F480

C

C

G

G

DC DG

DC DG

102

102

103

103

104

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

105

102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105

102 103 104 105 102 103 104 105 102 103 104 105

169 159

Ly6G Ly6G Ly6G Ly6G

CD19

CD3120576 CD3120576 CD3120576 CD3120576

Blood leukocytes

(a)

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105

C G DC DG

CD3120576 CD3120576 CD3120576 CD3120576

CD19

456 463 332 348

MLN cells

(b)

102 103 104 105

Gated cells

Gated cells

Gated cells

FSC

FSC

CD4 or CD8

PSGL-1 12057241205737

12057241205737

CD11a CCR9

CCR9

Cou

nt

Cou

nt

(c)

Figure 2 Representative flow cytometry plots Blood leukocytes (a) were defined by gating on CD45-positive cells The percentage of Ly6G-positive neutrophils from an individual representative mouse per group is listed For analyzing the lymphocyte population in MLNs (b)MLN cells were first gated to exclude debris Numbers indicate the percentage of CD3120576-positive lymphocytes in MLN cells For analyzingthe expression of adhesion molecules and chemokine receptors by T cells (c) lymphocytes were first identified based on low FSC and SSCcharacteristics CD4- or CD8-positive lymphocytes were gated to analyze the percentages of adhesion molecule- and chemokine receptor-expressing CD4+ and CD8+ lymphocytes Representative dot plots of leukocytes in blood are shown

Mediators of Inflammation 5

0 1 2 3 4 5 6 7 8 9 10

Body

wei

ght (

g)

0

18

20

22

24

26

28

30

CG

DC

Days

DG

daggerlowast

lowast

(a)

C G DC DG

Col

on w

eigh

tlen

gth

ratio

(gc

m)

000

002

004

006

008

010

daggerlowast

lowast

(b)

Figure 3 Body weight (a) and weightlength ratio of the colon (b) Data are presented as the mean plusmn SEM C normal mice fed the controldiet G normal mice fed a glutamine-enriched diet DC DSS group fed the control diet DG DSS group fed a glutamine-enriched dietlowastSignificantly different from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

The weightlength ratio of the colon an indicator of colonicedema was significantly higher in the colitis groups than theC group (Figure 3(b)) Treatment withGln attenuated colonicedema associated with DSS-induced inflammation

32 Leukocyte Populations in Blood and MLNs There wasno significant difference in blood or MLN leukocyte sub-populations between the C and G groups Compared to theC group the DSS colitis groups had a higher percentage ofblood neutrophils and lower T-cell population inMLNs DSSexposure did not alter the blood monocyte and lymphocytedistributions Also subsets of effector T cells in the blood andMLNs did not change Gln supplementation had no influenceon blood leukocyte or MLN lymphocyte populations innormal or colitic mice (Table 2)

33 Adhesion Molecule and CCR9 Expressions by Th CellsPercentages of adhesion molecules and CCR9 expressed byblood and MLN Th cells did not differ between the C and Ggroups DSS administration resulted in higher PSGL-1 CD11a(LFA-1 120572L subunit) and CCR9 expressions byThcells in bothblood and MLNs whereas no difference in 12057241205737 integrinexpression was detected among the control and DSS groupsMice in the DG group had lower percentages of PSGL-1-CD11a- and CCR9-expressing Th cells in blood (Figures4(a)ndash4(d)) and MLNs (Figures 5(a)ndash5(d)) The expressionlevel of CCR9 on 12057241205737-positive Th cells was also suppressedin the DG group (Figures 4(e) and 5(e))

34 Adhesion Molecule and CCR9 Expressions by Tc CellsNo differences in adhesion molecules and CCR9 expressedby blood and MLN Tc cells were observed between the Cand G groupsThe DSS colitis groups had higher percentages

Table 2 Leukocyte populations in blood and mesenteric lymphnodes (MLNs) ()

C G DC DGBlood

Neutrophils 87 plusmn 05 80 plusmn 09 161 plusmn 27lowast 169 plusmn 21lowast

Monocytes 72 plusmn 09 67 plusmn 07 82 plusmn 13 77 plusmn 15T cells 119 plusmn 04 117 plusmn 04 93 plusmn 12 93 plusmn 14

B cells 503 plusmn 09 534 plusmn 31 532 plusmn 27 490 plusmn 21

Th cells 67 plusmn 16 64 plusmn 04 73 plusmn 12 81 plusmn 08

Tc cells 36 plusmn 08 44 plusmn 03 45 plusmn 02 41 plusmn 03

MLNsT cells 452 plusmn 24 467 plusmn 22 321 plusmn 23

lowast

349 plusmn 10lowast

B cells 303 plusmn 18 290 plusmn 33 313 plusmn 21 297 plusmn 14

Th cells 118 plusmn 03 123 plusmn 07 112 plusmn 09 121 plusmn 09

Tc cells 63 plusmn 04 67 plusmn 06 58 plusmn 03 65 plusmn 05

CD45-positive cells were considered to be leukocytes and gated to determinethe population of leukocytes using a flow cytometer Staining for Ly-6G F480 CD3120576 and CD19 was used to respectively identify neutrophilmonocyte T cell and B cell populations For the analysis of T cell subpop-ulations lymphocytes were gated on the basis of their forward-and side-scatter profiles Percentages of T helper (Th) and cytotoxic T (Tc) cells wererespectively determined by CD4-and CD8-expressing cells in lymphocytesValues are presented as the mean plusmn SEM lowastSignificantly differs from the Cgroup (119875 lt 005)

of PSGL-1- CD11a- and CCR9-expressing blood and MLNTc cells whereas expression levels of 12057241205737 integrins did notdiffer among the 4 groups Compared to the DC group theDG group had lower expression of CD11a by blood (Figures6(a)ndash6(d)) and MLN Tc cells (Figures 7(a)ndash7(d)) There wasno difference in expression levels of CCR9 on 12057241205737-positiveTc cells between the 2 DSS colitis groups (Figures 6(e) and7(e))

6 Mediators of Inflammation

0

10

20

30

40

50

C G DC DG

PSG

L-1

()

lowast

lowast

dagger

(a)

0

2

4

6

8

10

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

5

10

15

20

25

C G DC DG

dagger

lowast

CD11

a (

)

(c)

0

10

20

30

40

50

C G DC DG

dagger

lowast

lowastCCR9

(

)

(d)

0

15

30

45

60

C G DC DG

lowast

CCR

9 ex

pres

sing

dagger

CD4+12057241205737+

T ce

lls (

)

(e)

Figure 4 Percentage of adhesionmolecule- and chemokine receptor-expressingThelper (Th) cells in blood CD4-positive blood lymphocyteswere gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d)) (e) Expression of CCR9 by CD4and 12057241205737 integrin double-positive blood lymphocytes Values are shown as the mean plusmn SEM lowastSignificantly different from the C group(119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

35 Gene Expression of Endothelial Adhesion Molecules inColon Tissues There was no difference in mRNA levels ofICAM-1 P-selectin and E-selectin between the C and Ggroups in colon tissues DSS-induced colitis greatly upreg-ulated the adhesion molecule genes expressed by activatedendothelial cells Compared to the DC group the expression

levels of ICAM-1 P-selectin and E-selectin mRNA weresuppressed in the DG group (Figure 8)

36 T Lymphocyte Subsets in the Colon Mucosa CD3 is thecell surface marker of T cells CD3 and CD4 double-positivecells are considered Th cells whereas Tc cells coexpress CD3

Mediators of Inflammation 7

0

10

20

30

40

50

C G DC DG

PSG

L-1

()

lowast

lowast

dagger

(a)

0

2

4

6

8

10

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

10

20

30

40

50

C G DC DG

CD11

a (

)

dagger

lowast

(c)

0

10

20

30

40

50

C G DC DG

dagger

lowast

CCR9

(

)

(d)

0

15

30

45

60

C G DC DG

dagger

lowast

CCR

9 ex

pres

sing

CD4+12057241205737+

T ce

lls (

)

(e)

Figure 5 Percentage of adhesion molecule- and chemokine receptor-expressing T helper (Th) cells in mesenteric lymph nodes (MLNs)CD4-positiveMLN lymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d))(e) Expression of CCR9 by CD4 and 12057241205737 integrin double-positive MLN lymphocytes Values are shown as the mean plusmn SEM lowastSignificantlydifferent from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

andCD8As shown in Figure 9 the immunoreactive intensityof Th cells was higher in the DC group than the DG groupHowever intensities of Tc cell populations did not differbetween the DC and DG groups (Figure 10)

4 Discussion

DSS is a heparin-like polysaccharide which results in acutechemical toxicity that disrupts the intestinal epithelial cell

8 Mediators of Inflammation

0

10

20

30

40

50

C G DC DG

lowast lowast

PSG

L-1

()

(a)

0

5

10

15

20

25

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

5

10

15

20

25

C G DC DG

dagger

lowast

CD11

a (

)

(c)

0

10

20

30

40

50

C G DC DG

CCR9

()

lowast

lowast

(d)

0

20

40

60

80

100

C G DC DG

lowastlowast

CCR

9 ex

pres

sing

CD8+12057241205737+

T ce

lls (

)

(e)

Figure 6 Percentage of adhesion molecule- and chemokine receptor-expressing cytotoxic T (Tc) cells in blood CD8-positive bloodlymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d)) (e) Expressionof CCR9 by CD8 and 12057241205737 integrin double-positive blood lymphocytes Values are shown as the mean plusmn SEM lowastSignificantly different fromthe C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

barrier [21] DSS-induced colitis is characterized by extensivecrypt and epithelial cell damage with ulceration tissueedema and infiltration of immune cells predominantly in thedistal colon that mimics the histological features of UC [22]Recent studies indicated that DSS-induced morphological

and biochemical damage also extends to the small intestines[23] Susceptibilities to DSS-induced colitis differ in variousinbred mouse strains [24] A single cycle of DSS exposureto the C57BL6 strain was found to develop acute colitiswhich later proceeds to chronic inflammation and T cell

Mediators of Inflammation 9

C G DC DG

PSG

L-1

()

0

10

20

30

40

50

lowastlowast

(a)

CD11

a (

)

C G DC DG0

5

10

15

20

25

CD11

a (

)

12057241205737

inte

grin

s (

)

(b)

C G DC DG0

5

10

15

20

25

CD11

a (

)

dagger

lowast

(c)

CCR9

()

C G DC DG0

5

10

15

20

25

lowast

lowast

(d)

CCR

9 ex

pres

sing

0

15

30

45

60

lowastlowast

CD+ 812057241205737+

T ce

lls (

)

C G DC DG

(e)

Figure 7 Percentage of adhesion molecule- and chemokine receptor-expressing cytotoxic T (Tc) cells in mesenteric lymph nodes (MLNs)CD8-positiveMLN lymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d))(e) Expression of CCR9 by CD8 and 12057241205737 integrin double-positive MLN lymphocytes Values are shown as the mean plusmn SEM lowastSignificantlydifferent from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

migration to the colon plays an important role in theprogression to chronicity [25] Regarding the high sensitivityto DSS C57BL6 mice were used in this study to analyze theconsequences of DSS exposure on adhesion molecules and

chemokine receptors involved in T cell trafficking to theintestines

IBD is associated with a massive influx of immune cellsinto the gut Previous studies indicated that increased local

10 Mediators of Inflammation

ICAM-1 P-selectin E-selectin

Relat

ive m

RNA

ratio

0

50

100

150

200

1000

1250

1500

CG

DCDG

daggerdagger

dagger

lowast

lowastlowast

lowast

lowast

lowast

Figure 8 Gene expression of endothelial adhesion molecules incolon tissues mRNA levels were analyzed by a real-time PCR TheC group was used as a calibrator and the data were presented as thefold change in gene expression relative to the calibrator Values areshown as themean plusmn SEM lowastSignificantly different from the C group(119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

secretion of proinflammatory cytokines by the inflamedcolon leads to upregulated expressions of vascular adhesionmolecules resulting in a sustained influx of inflammatorycells [26] Although inflammation predominantly occurs inthe GI tract IBD patients are likely to develop extraintestinalmanifestations [27] that may be attributed to aberrant acti-vation and homing of T cells [28] A recent study indicatedthat circulating CD4+ and CD8+ T cells are activated in bothCD and UC patients and these cells were correlated withleakage of microbial products from the impaired intestinalbarrier [5] Also the severity of DSS-induced colitis wascorrelatedwith the immune response ofMLNs [29] A similarphenomenon was also observed in this study Our resultsindicated that expressions of adhesion molecules and CCR9on Th cells and Tc cells significantly increased in both bloodand MLNs after DSS exposure These findings suggest thatboth local and systemic T cells are activated The role of Thcells in the pathogenesis of IBD has been widely studiedDysregulation ofTh cells can lead to immune cell infiltrationinto the intestinal mucosa and cause persistent inflammation[3 6] The pathogenic role of Tc cells in IBD has been lessinvestigated A previous study showed that antigen-specificTc cells caused relapsing colitis in normal mice due to thecytolytic function against the intestinal epithelium [30] Leeet al [4] reported a gene expression profile of circulatingCD8+ T cells that predicted a more aggressive disease coursefor IBD patients However the modulatory mechanism of Tccells in IBD is still under investigation

Naıve T cells constantly recirculate between the bloodand secondary lymphoid organs Once activated in secondarylymphoid organs they become effector T cells that expressadhesion molecules and chemokine receptors which controltheir extravasation into nonlymphoid tissue sites [31] T cell

trafficking to the gut and gut-associated lymphoid tissues(GALTs) requires 12057241205737 integrins The ligand MAdCAM-1is constitutively expressed by the mucosal endothelium inthe small intestine and colon [32] CCR9 is thought toparticipate in the specific localization of T cells to the smallintestines because the sole ligand for CCR9 C-C chemokineligand 25 (CCL25) is strongly expressed by the small intesti-nal epithelium [33] However Wurbel et al [34] revealedthat CCL25 expression increased during the recovery phaseafter acute DSS administration suggesting a regulatory roleof CCR9CCL25 interactions during colonic inflammationIn this study percentages of PSGL-1- LFA-1- and CCR9-expressing T cells were upregulated in acute DSS colitiswhereas expression levels of 12057241205737 integrins in colitic micedid not differ from those of normal mice It is possible thatT cell trafficking into the gut during acute DSS exposure isless dependent on 12057241205737 integrins In support of our findingsWang et al [35] reported that localization of T cells to theintestines was relatively unaffected by 12057241205737 blockade duringacute DSS-induced colitis

Gln is a critical fuel source for enterocytes and immunecells Gln supplementation attenuates gut injury by a com-plex mechanism which involves protecting the epithelialbarrier function reducing oxidative stress and modulat-ing inflammatory responses [36] The local and systemicimmunomodulatory effects of Gln have been discussed invarious experimental colitis models via different adminis-tration routes Studies using rodents with trinitrobenzenesulfonic acid-induced colitis indicated that Gln given bythe rectal route inhibits nuclear factor (NF)-120581B- and STAT-mediated inflammation in colon tissues [15] and further pre-vents colon fibrosis through downregulating gene pathwaysthat contribute to the accumulation of matrix proteins [37]We recently demonstrated that intraperitoneal pretreatmentwith alanyl-Gln a Gln-containing dipeptide widely used inparenteral nutrition suppresses cytokine expression in bloodTh cells reduces NF-120581B-mediated inflammatory responsesin the colon and upregulates expressions of genes whichpromote recovery of the colonic mucosa [17 38]

Because most exogenous Gln is absorbed in the proximalsmall intestine it might not reach the inflamed colon at asufficient concentration tomodulate inflammatory responses[36] However the enteral route of Gln administrationstill showed protective effects against DSS-induced damageOral Gln supplementation reduced the feces water contentenhanced expression of HSPs in the colonic mucosa andameliorated colon injury caused byDSS exposure [16 39 40]A previous study indicated that oral Gln attenuated leukocyteadhesion and emigration in a rodentmodel of indomethacin-induced ileitis [41] In this study we demonstrated thatoral Gln administration suppressed adhesion molecules andCCR9 expressed by T cells and downregulated the mRNAlevels of adhesion molecules expressed by endothelium incolon tissues The histological findings also support theresults that Gln administration suppressed the infiltrationof Th cells into the colon mucosa Gln consumption is animportant component of T cell activation [42 43] Differentsusceptibilities ofThandTc cells toGln supplementationmaybe explained by a higher cell population of Th cells which

Mediators of Inflammation 11

50120583m

50120583m

50120583m

50120583m

CD3

C

G

DC

DG

(a)

50120583m

50120583m

50120583m

50120583m

CD4

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 9 Representative examples of the immunofluorescence staining of T helper cells In the left column cell surface antigens were stainedfor CD3 (green FITC) and staining of CD4 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI(blue) and the last column presents a colocalized fluorescence image of CD3+CD4+ T cells

competes as a Gln source with Tc cells Further studies areneeded to investigate the molecular mechanisms involved ingene expressions of adhesion molecules and CCR9 regulatedby Gln

In conclusion this study showed for the first time thatpretreatment with oral Gln reduced adhesion molecule- andCCR9-expressing T cells induced by DSS exposure Theinhibitory abilities against adhesion molecule and CCR9expressions were more obvious inTh cells than Tc cells AlsoGln supplementation reduced gene expressions of endothe-lial adhesion molecules in colons prevented BW loss and

attenuated colon edema in coliticmice Our results imply thatdietary Gln prevented Th cell trafficking into colon tissuesand provide a new mechanism of Gln supplementation thathas beneficial effects on ameliorating the severity of acuteDSS-induced colitis

Conflict of Interests

The authors declare that they have no conflict of interests

12 Mediators of Inflammation

50120583m

CD3

50120583m

50120583m

50120583m

C

G

DC

DG

(a)

CD8

50120583m

50120583m

50120583m

50120583m

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 10 Representative immunofluorescence images of cytotoxic T cells In the left column cell surface antigens were stained for CD3(green FITC) and the staining of CD8 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI (blue)and the last column presents a colocalized fluorescence image of CD3+CD8+ T cells

Acknowledgments

The authors would like to thank Man-Hui Pai Assistant Pro-fessor of Department of Anatomy Taipei Medical Universityfor her technical assistance in immunofluorescence stainingThis studywas supported by aResearchGrant (NSC100-2320-B-038-007-MY3) from the National Science Council TaipeiTaiwan

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] C T Murphy K Nally F Shanahan and S Melgar ldquoShining alight on intestinal trafficrdquoClinical and Developmental Immunol-ogy vol 2012 Article ID 808157 14 pages 2012

Mediators of Inflammation 13

[3] L A Zenewicz A Antov and R A Flavell ldquoCD4 T-cell differ-entiation and inflammatory bowel diseaserdquo Trends inMolecularMedicine vol 15 no 5 pp 199ndash207 2009

[4] J C Lee P A Lyons E F McKinney et al ldquoGene expressionprofiling of CD8+ T cells predicts prognosis in patients withCrohn disease and ulcerative colitisrdquo Journal of Clinical Inves-tigation vol 121 no 10 pp 4170ndash4179 2011

[5] N T Funderburg S R Stubblefield Park H C Sung et alldquoCirculating CD4+ and CD8+ T cells are activated in IBDand are associated with plasma markers of inflammationrdquoImmunology vol 140 no 1 pp 87ndash97 2013

[6] E J Villablanca B Cassani U H von Andrian and J RMora ldquoBlocking lymphocyte localization to the gastrointestinalmucosa as a therapeutic strategy for inflammatory boweldiseasesrdquo Gastroenterology vol 140 no 6 pp 1776ndash1784 2011

[7] R P McEver and R D Cummings ldquoPerspectives series celladhesion in vascular biology Role of PSGL-1 binding toselectins in leukocyte recruitmentrdquo The Journal of ClinicalInvestigation vol 100 no 3 pp 485ndash491 1997

[8] C Berlin E L Berg M J Briskin et al ldquo12057241205737 Integrin medi-ates lymphocyte binding to the mucosal vascular addressinMAdCAM-1rdquo Cell vol 74 no 1 pp 185ndash195 1993

[9] K Ley C Laudanna M I Cybulsky and S Nourshargh ldquoGet-ting to the site of inflammation the leukocyte adhesion cascadeupdatedrdquoNature Reviews Immunology vol 7 no 9 pp 678ndash6892007

[10] B Moser M Wolf A Walz and P Loetscher ldquoChemokinesmultiple levels of leukocyte migration controlrdquo Trends inImmunology vol 25 no 2 pp 75ndash84 2004

[11] S Arimilli W Ferlin N Solvason S Deshpande M Howardand S Mocci ldquoChemokines in autoimmune diseasesrdquo Immuno-logical Reviews vol 177 pp 43ndash51 2000

[12] C Koenecke and R Forster ldquoCCR9 and inflammatory boweldiseaserdquo Expert Opinion on Therapeutic Targets vol 13 no 3pp 297ndash306 2009

[13] B Johansson-Lindbom and W W Agace ldquoGeneration of gut-homing T cells and their localization to the small intestinalmucosardquo Immunological Reviews vol 215 no 1 pp 226ndash2422007

[14] P EWischmeyer ldquoGlutaminemode of action in critical illnessrdquoCritical Care Medicine vol 35 no 9 pp S541ndashS544 2007

[15] N A Kretzmann H Fillmann J L Mauriz C A MarroniJ Gonzalez-Gallego and M J Tunon ldquoEffects of glutamineon proinflammatory gene expression and activation of nuclearfactor Kappa B and signal transducers and activators oftranscription in TNBS-induced colitisrdquo Inflammatory BowelDiseases vol 14 no 11 pp 1504ndash1513 2008

[16] H Xue A J D Sufit and P EWischmeyer ldquoGlutamine therapyimproves outcome of in vitro and in vivo experimental colitismodelsrdquo Journal of Parenteral and Enteral Nutrition vol 35 no2 pp 188ndash197 2011

[17] C-C Chu Y-C Hou M-H Pai C-J Chao and S-L YehldquoPretreatment with alanyl-glutamine suppresses T-helper-cell-associated cytokine expression and reduces inflammatoryresponses in mice with acute DSS-induced colitisrdquo The Journalof Nutritional Biochemistry vol 23 no 9 pp 1092ndash1099 2011

[18] S Kew S M Wells P Yaqoob F A Wallace E A Miles and PC Calder ldquoDietary glutamine enhances murine T-lymphocyteresponsivenessrdquo The Journal of Nutrition vol 129 no 8 pp1524ndash1531 1999

[19] S M Wells S Kew P Yaqoob F A Wallace and P C CalderldquoDietary glutamine enhances cytokine production by murinemacrophagesrdquo Nutrition vol 15 no 11-12 pp 881ndash884 1999

[20] Y-C Hou M-H Pai W-C Chiu Y-M Hu and S-L YehldquoEffects of dietary glutamine supplementation on lung injuryinduced by lipopolysaccharide administrationrdquo American Jour-nal of PhysiologymdashLung Cellular and Molecular Physiology vol296 no 3 pp L288ndashL295 2009

[21] M Kawada A Arihiro and E Mizoguchi ldquoInsights fromadvances in research of chemically induced experimental mod-els of human inflammatory bowel diseaserdquo World Journal ofGastroenterology vol 13 no 42 pp 5581ndash5593 2007

[22] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[23] R Yazbeck G S Howarth R N Butler M S Geier and CA Abbott ldquoBiochemical and histological changes in the smallintestine of mice with dextran sulfate sodium colitisrdquo Journal ofCellular Physiology vol 226 no 12 pp 3219ndash3224 2011

[24] M Mahler I J Bristol E H Leiter et al ldquoDifferentialsusceptibility of inbred mouse strains to dextran sulfatesodium-induced colitisrdquo American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 274 no 3 part 1 ppG544ndashG551 1998

[25] S Melgar A Karlsson and E Michaelsson ldquoAcute colitisinduced by dextran sulfate sodium progresses to chronicityin C57BL6 but not in BALBc mice correlation betweensymptoms and inflammationrdquoAmerican Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 288 no 6 pp G1328ndashG1338 2005

[26] MKoizumiN King R Lobb C Benjamin andDK PodolskyldquoExpression of vascular adhesion molecules in inflammatorybowel diseaserdquo Gastroenterology vol 103 no 3 pp 840ndash8471992

[27] L Peyrin-Biroulet E V Loftus Jr J-F Colombel and W JSandborn ldquoLong-term complications extraintestinal manifes-tations and mortality in adult Crohnrsquos disease in population-based cohortsrdquo Inflammatory Bowel Diseases vol 17 no 1 pp471ndash478 2011

[28] D H Adams and B Eksteen ldquoAberrant homing of mucosal Tcells and extra-intestinal manifestations of inflammatory boweldiseaserdquoNature Reviews Immunology vol 6 no 3 pp 244ndash2512006

[29] TW Spahn H Herbst P D Rennert et al ldquoInduction of colitisinmice deficient of Peyerrsquos patches andmesenteric lymph nodesis associated with increased disease severity and formation ofcolonic lymphoid patchesrdquo The American Journal of Pathologyvol 161 no 6 pp 2273ndash2282 2002

[30] S Nancey S Holvoet I Graber et al ldquoCD8+ Cytotoxic T CellsInduce Relapsing Colitis in Normal Micerdquo Gastroenterologyvol 131 no 2 pp 485ndash496 2006

[31] E C Butcher and L J Picker ldquoLymphocyte homing and home-ostasisrdquo Science vol 272 no 5258 pp 60ndash66 1996

[32] C Berlin R F Bargatze J J Campbell et al ldquo1205724 Integrinsmediate lymphocyte attachment and rolling under physiologicflowrdquo Cell vol 80 no 3 pp 413ndash422 1995

[33] K A Papadakis J Prehn V Nelson et al ldquoThe role of thymus-expressed chemokine and its receptor CCR9 on lymphocytesin the regional specialization of the mucosal immune systemrdquoJournal of Immunology vol 165 no 9 pp 5069ndash5076 2000

14 Mediators of Inflammation

[34] M-A Wurbel M G McIntire P Dwyer and E FiebigerldquoCCL25CCR9 interactions regulate large intestinal inflamma-tion in a murine model of acute colitisrdquo PLoS ONE vol 6 no 1Article ID e16442 2011

[35] CWang E K Hanly LWWheeler M Kaur K GMcDonaldand R D Newberry ldquoEffect of 12057241205737 blockade on intestinallymphocyte subsets and lymphoid tissue developmentrdquo Inflam-matory Bowel Diseases vol 16 no 10 pp 1751ndash1762 2010

[36] M Coeffier R Marion-Letellier and P Dechelotte ldquoPotentialfor amino acids supplementation during inflammatory boweldiseasesrdquo Inflammatory Bowel Diseases vol 16 no 3 pp 518ndash524 2010

[37] B San-Miguel I Crespo N A Kretzmann et al ldquoGlutamineprevents fibrosis development in rats with colitis induced by246-trinitrobenzene sulfonic acidrdquo The Journal of Nutritionvol 140 no 6 pp 1065ndash1071 2010

[38] Y C Hou C C Chu T L Ko et al ldquoEffects of alanyl-glutaminedipeptide on the expression of colon-inflammatory mediatorsduring the recovery phase of colitis induced by dextran sulfatesodiumrdquo European Journal of Nutrition vol 52 no 3 pp 1089ndash1098 2013

[39] Y Shiomi S Nishiumi M Ooi et al ldquoGCMS-based meta-bolomic study in mice with colitis induced by dextran sulfatesodiumrdquo Inflammatory Bowel Diseases vol 17 no 11 pp 2261ndash2274 2011

[40] M Vicario C Amat M Rivero M Moreto and C PelegrıldquoDietary glutamine affects mucosal functions in rats with mildDSS-induced colitisrdquoThe Journal of Nutrition vol 137 no 8 pp1931ndash1937 2007

[41] H Arndt F Kullmann F Reuszlig J Scholmerich and K-D Pal-itzsch ldquoGlutamine attenuates leukocyte-endothelial cell adhe-sion in indomethacin-induced intestinal inflammation in theratrdquo Journal of Parenteral and Enteral Nutrition vol 23 no 1pp 12ndash18 1999

[42] P Yaqoob and P C Calder ldquoGlutamine requirement of prolif-erating T lymphocytesrdquo Nutrition vol 13 no 7-8 pp 646ndash6511997

[43] E L Carr A Kelman G S Wu et al ldquoGlutamine uptake andmetabolism are coordinately regulated by ERKMAPK duringT lymphocyte activationrdquo Journal of Immunology vol 185 no2 pp 1037ndash1044 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 5

0 1 2 3 4 5 6 7 8 9 10

Body

wei

ght (

g)

0

18

20

22

24

26

28

30

CG

DC

Days

DG

daggerlowast

lowast

(a)

C G DC DG

Col

on w

eigh

tlen

gth

ratio

(gc

m)

000

002

004

006

008

010

daggerlowast

lowast

(b)

Figure 3 Body weight (a) and weightlength ratio of the colon (b) Data are presented as the mean plusmn SEM C normal mice fed the controldiet G normal mice fed a glutamine-enriched diet DC DSS group fed the control diet DG DSS group fed a glutamine-enriched dietlowastSignificantly different from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

The weightlength ratio of the colon an indicator of colonicedema was significantly higher in the colitis groups than theC group (Figure 3(b)) Treatment withGln attenuated colonicedema associated with DSS-induced inflammation

32 Leukocyte Populations in Blood and MLNs There wasno significant difference in blood or MLN leukocyte sub-populations between the C and G groups Compared to theC group the DSS colitis groups had a higher percentage ofblood neutrophils and lower T-cell population inMLNs DSSexposure did not alter the blood monocyte and lymphocytedistributions Also subsets of effector T cells in the blood andMLNs did not change Gln supplementation had no influenceon blood leukocyte or MLN lymphocyte populations innormal or colitic mice (Table 2)

33 Adhesion Molecule and CCR9 Expressions by Th CellsPercentages of adhesion molecules and CCR9 expressed byblood and MLN Th cells did not differ between the C and Ggroups DSS administration resulted in higher PSGL-1 CD11a(LFA-1 120572L subunit) and CCR9 expressions byThcells in bothblood and MLNs whereas no difference in 12057241205737 integrinexpression was detected among the control and DSS groupsMice in the DG group had lower percentages of PSGL-1-CD11a- and CCR9-expressing Th cells in blood (Figures4(a)ndash4(d)) and MLNs (Figures 5(a)ndash5(d)) The expressionlevel of CCR9 on 12057241205737-positive Th cells was also suppressedin the DG group (Figures 4(e) and 5(e))

34 Adhesion Molecule and CCR9 Expressions by Tc CellsNo differences in adhesion molecules and CCR9 expressedby blood and MLN Tc cells were observed between the Cand G groupsThe DSS colitis groups had higher percentages

Table 2 Leukocyte populations in blood and mesenteric lymphnodes (MLNs) ()

C G DC DGBlood

Neutrophils 87 plusmn 05 80 plusmn 09 161 plusmn 27lowast 169 plusmn 21lowast

Monocytes 72 plusmn 09 67 plusmn 07 82 plusmn 13 77 plusmn 15T cells 119 plusmn 04 117 plusmn 04 93 plusmn 12 93 plusmn 14

B cells 503 plusmn 09 534 plusmn 31 532 plusmn 27 490 plusmn 21

Th cells 67 plusmn 16 64 plusmn 04 73 plusmn 12 81 plusmn 08

Tc cells 36 plusmn 08 44 plusmn 03 45 plusmn 02 41 plusmn 03

MLNsT cells 452 plusmn 24 467 plusmn 22 321 plusmn 23

lowast

349 plusmn 10lowast

B cells 303 plusmn 18 290 plusmn 33 313 plusmn 21 297 plusmn 14

Th cells 118 plusmn 03 123 plusmn 07 112 plusmn 09 121 plusmn 09

Tc cells 63 plusmn 04 67 plusmn 06 58 plusmn 03 65 plusmn 05

CD45-positive cells were considered to be leukocytes and gated to determinethe population of leukocytes using a flow cytometer Staining for Ly-6G F480 CD3120576 and CD19 was used to respectively identify neutrophilmonocyte T cell and B cell populations For the analysis of T cell subpop-ulations lymphocytes were gated on the basis of their forward-and side-scatter profiles Percentages of T helper (Th) and cytotoxic T (Tc) cells wererespectively determined by CD4-and CD8-expressing cells in lymphocytesValues are presented as the mean plusmn SEM lowastSignificantly differs from the Cgroup (119875 lt 005)

of PSGL-1- CD11a- and CCR9-expressing blood and MLNTc cells whereas expression levels of 12057241205737 integrins did notdiffer among the 4 groups Compared to the DC group theDG group had lower expression of CD11a by blood (Figures6(a)ndash6(d)) and MLN Tc cells (Figures 7(a)ndash7(d)) There wasno difference in expression levels of CCR9 on 12057241205737-positiveTc cells between the 2 DSS colitis groups (Figures 6(e) and7(e))

6 Mediators of Inflammation

0

10

20

30

40

50

C G DC DG

PSG

L-1

()

lowast

lowast

dagger

(a)

0

2

4

6

8

10

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

5

10

15

20

25

C G DC DG

dagger

lowast

CD11

a (

)

(c)

0

10

20

30

40

50

C G DC DG

dagger

lowast

lowastCCR9

(

)

(d)

0

15

30

45

60

C G DC DG

lowast

CCR

9 ex

pres

sing

dagger

CD4+12057241205737+

T ce

lls (

)

(e)

Figure 4 Percentage of adhesionmolecule- and chemokine receptor-expressingThelper (Th) cells in blood CD4-positive blood lymphocyteswere gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d)) (e) Expression of CCR9 by CD4and 12057241205737 integrin double-positive blood lymphocytes Values are shown as the mean plusmn SEM lowastSignificantly different from the C group(119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

35 Gene Expression of Endothelial Adhesion Molecules inColon Tissues There was no difference in mRNA levels ofICAM-1 P-selectin and E-selectin between the C and Ggroups in colon tissues DSS-induced colitis greatly upreg-ulated the adhesion molecule genes expressed by activatedendothelial cells Compared to the DC group the expression

levels of ICAM-1 P-selectin and E-selectin mRNA weresuppressed in the DG group (Figure 8)

36 T Lymphocyte Subsets in the Colon Mucosa CD3 is thecell surface marker of T cells CD3 and CD4 double-positivecells are considered Th cells whereas Tc cells coexpress CD3

Mediators of Inflammation 7

0

10

20

30

40

50

C G DC DG

PSG

L-1

()

lowast

lowast

dagger

(a)

0

2

4

6

8

10

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

10

20

30

40

50

C G DC DG

CD11

a (

)

dagger

lowast

(c)

0

10

20

30

40

50

C G DC DG

dagger

lowast

CCR9

(

)

(d)

0

15

30

45

60

C G DC DG

dagger

lowast

CCR

9 ex

pres

sing

CD4+12057241205737+

T ce

lls (

)

(e)

Figure 5 Percentage of adhesion molecule- and chemokine receptor-expressing T helper (Th) cells in mesenteric lymph nodes (MLNs)CD4-positiveMLN lymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d))(e) Expression of CCR9 by CD4 and 12057241205737 integrin double-positive MLN lymphocytes Values are shown as the mean plusmn SEM lowastSignificantlydifferent from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

andCD8As shown in Figure 9 the immunoreactive intensityof Th cells was higher in the DC group than the DG groupHowever intensities of Tc cell populations did not differbetween the DC and DG groups (Figure 10)

4 Discussion

DSS is a heparin-like polysaccharide which results in acutechemical toxicity that disrupts the intestinal epithelial cell

8 Mediators of Inflammation

0

10

20

30

40

50

C G DC DG

lowast lowast

PSG

L-1

()

(a)

0

5

10

15

20

25

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

5

10

15

20

25

C G DC DG

dagger

lowast

CD11

a (

)

(c)

0

10

20

30

40

50

C G DC DG

CCR9

()

lowast

lowast

(d)

0

20

40

60

80

100

C G DC DG

lowastlowast

CCR

9 ex

pres

sing

CD8+12057241205737+

T ce

lls (

)

(e)

Figure 6 Percentage of adhesion molecule- and chemokine receptor-expressing cytotoxic T (Tc) cells in blood CD8-positive bloodlymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d)) (e) Expressionof CCR9 by CD8 and 12057241205737 integrin double-positive blood lymphocytes Values are shown as the mean plusmn SEM lowastSignificantly different fromthe C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

barrier [21] DSS-induced colitis is characterized by extensivecrypt and epithelial cell damage with ulceration tissueedema and infiltration of immune cells predominantly in thedistal colon that mimics the histological features of UC [22]Recent studies indicated that DSS-induced morphological

and biochemical damage also extends to the small intestines[23] Susceptibilities to DSS-induced colitis differ in variousinbred mouse strains [24] A single cycle of DSS exposureto the C57BL6 strain was found to develop acute colitiswhich later proceeds to chronic inflammation and T cell

Mediators of Inflammation 9

C G DC DG

PSG

L-1

()

0

10

20

30

40

50

lowastlowast

(a)

CD11

a (

)

C G DC DG0

5

10

15

20

25

CD11

a (

)

12057241205737

inte

grin

s (

)

(b)

C G DC DG0

5

10

15

20

25

CD11

a (

)

dagger

lowast

(c)

CCR9

()

C G DC DG0

5

10

15

20

25

lowast

lowast

(d)

CCR

9 ex

pres

sing

0

15

30

45

60

lowastlowast

CD+ 812057241205737+

T ce

lls (

)

C G DC DG

(e)

Figure 7 Percentage of adhesion molecule- and chemokine receptor-expressing cytotoxic T (Tc) cells in mesenteric lymph nodes (MLNs)CD8-positiveMLN lymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d))(e) Expression of CCR9 by CD8 and 12057241205737 integrin double-positive MLN lymphocytes Values are shown as the mean plusmn SEM lowastSignificantlydifferent from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

migration to the colon plays an important role in theprogression to chronicity [25] Regarding the high sensitivityto DSS C57BL6 mice were used in this study to analyze theconsequences of DSS exposure on adhesion molecules and

chemokine receptors involved in T cell trafficking to theintestines

IBD is associated with a massive influx of immune cellsinto the gut Previous studies indicated that increased local

10 Mediators of Inflammation

ICAM-1 P-selectin E-selectin

Relat

ive m

RNA

ratio

0

50

100

150

200

1000

1250

1500

CG

DCDG

daggerdagger

dagger

lowast

lowastlowast

lowast

lowast

lowast

Figure 8 Gene expression of endothelial adhesion molecules incolon tissues mRNA levels were analyzed by a real-time PCR TheC group was used as a calibrator and the data were presented as thefold change in gene expression relative to the calibrator Values areshown as themean plusmn SEM lowastSignificantly different from the C group(119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

secretion of proinflammatory cytokines by the inflamedcolon leads to upregulated expressions of vascular adhesionmolecules resulting in a sustained influx of inflammatorycells [26] Although inflammation predominantly occurs inthe GI tract IBD patients are likely to develop extraintestinalmanifestations [27] that may be attributed to aberrant acti-vation and homing of T cells [28] A recent study indicatedthat circulating CD4+ and CD8+ T cells are activated in bothCD and UC patients and these cells were correlated withleakage of microbial products from the impaired intestinalbarrier [5] Also the severity of DSS-induced colitis wascorrelatedwith the immune response ofMLNs [29] A similarphenomenon was also observed in this study Our resultsindicated that expressions of adhesion molecules and CCR9on Th cells and Tc cells significantly increased in both bloodand MLNs after DSS exposure These findings suggest thatboth local and systemic T cells are activated The role of Thcells in the pathogenesis of IBD has been widely studiedDysregulation ofTh cells can lead to immune cell infiltrationinto the intestinal mucosa and cause persistent inflammation[3 6] The pathogenic role of Tc cells in IBD has been lessinvestigated A previous study showed that antigen-specificTc cells caused relapsing colitis in normal mice due to thecytolytic function against the intestinal epithelium [30] Leeet al [4] reported a gene expression profile of circulatingCD8+ T cells that predicted a more aggressive disease coursefor IBD patients However the modulatory mechanism of Tccells in IBD is still under investigation

Naıve T cells constantly recirculate between the bloodand secondary lymphoid organs Once activated in secondarylymphoid organs they become effector T cells that expressadhesion molecules and chemokine receptors which controltheir extravasation into nonlymphoid tissue sites [31] T cell

trafficking to the gut and gut-associated lymphoid tissues(GALTs) requires 12057241205737 integrins The ligand MAdCAM-1is constitutively expressed by the mucosal endothelium inthe small intestine and colon [32] CCR9 is thought toparticipate in the specific localization of T cells to the smallintestines because the sole ligand for CCR9 C-C chemokineligand 25 (CCL25) is strongly expressed by the small intesti-nal epithelium [33] However Wurbel et al [34] revealedthat CCL25 expression increased during the recovery phaseafter acute DSS administration suggesting a regulatory roleof CCR9CCL25 interactions during colonic inflammationIn this study percentages of PSGL-1- LFA-1- and CCR9-expressing T cells were upregulated in acute DSS colitiswhereas expression levels of 12057241205737 integrins in colitic micedid not differ from those of normal mice It is possible thatT cell trafficking into the gut during acute DSS exposure isless dependent on 12057241205737 integrins In support of our findingsWang et al [35] reported that localization of T cells to theintestines was relatively unaffected by 12057241205737 blockade duringacute DSS-induced colitis

Gln is a critical fuel source for enterocytes and immunecells Gln supplementation attenuates gut injury by a com-plex mechanism which involves protecting the epithelialbarrier function reducing oxidative stress and modulat-ing inflammatory responses [36] The local and systemicimmunomodulatory effects of Gln have been discussed invarious experimental colitis models via different adminis-tration routes Studies using rodents with trinitrobenzenesulfonic acid-induced colitis indicated that Gln given bythe rectal route inhibits nuclear factor (NF)-120581B- and STAT-mediated inflammation in colon tissues [15] and further pre-vents colon fibrosis through downregulating gene pathwaysthat contribute to the accumulation of matrix proteins [37]We recently demonstrated that intraperitoneal pretreatmentwith alanyl-Gln a Gln-containing dipeptide widely used inparenteral nutrition suppresses cytokine expression in bloodTh cells reduces NF-120581B-mediated inflammatory responsesin the colon and upregulates expressions of genes whichpromote recovery of the colonic mucosa [17 38]

Because most exogenous Gln is absorbed in the proximalsmall intestine it might not reach the inflamed colon at asufficient concentration tomodulate inflammatory responses[36] However the enteral route of Gln administrationstill showed protective effects against DSS-induced damageOral Gln supplementation reduced the feces water contentenhanced expression of HSPs in the colonic mucosa andameliorated colon injury caused byDSS exposure [16 39 40]A previous study indicated that oral Gln attenuated leukocyteadhesion and emigration in a rodentmodel of indomethacin-induced ileitis [41] In this study we demonstrated thatoral Gln administration suppressed adhesion molecules andCCR9 expressed by T cells and downregulated the mRNAlevels of adhesion molecules expressed by endothelium incolon tissues The histological findings also support theresults that Gln administration suppressed the infiltrationof Th cells into the colon mucosa Gln consumption is animportant component of T cell activation [42 43] Differentsusceptibilities ofThandTc cells toGln supplementationmaybe explained by a higher cell population of Th cells which

Mediators of Inflammation 11

50120583m

50120583m

50120583m

50120583m

CD3

C

G

DC

DG

(a)

50120583m

50120583m

50120583m

50120583m

CD4

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 9 Representative examples of the immunofluorescence staining of T helper cells In the left column cell surface antigens were stainedfor CD3 (green FITC) and staining of CD4 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI(blue) and the last column presents a colocalized fluorescence image of CD3+CD4+ T cells

competes as a Gln source with Tc cells Further studies areneeded to investigate the molecular mechanisms involved ingene expressions of adhesion molecules and CCR9 regulatedby Gln

In conclusion this study showed for the first time thatpretreatment with oral Gln reduced adhesion molecule- andCCR9-expressing T cells induced by DSS exposure Theinhibitory abilities against adhesion molecule and CCR9expressions were more obvious inTh cells than Tc cells AlsoGln supplementation reduced gene expressions of endothe-lial adhesion molecules in colons prevented BW loss and

attenuated colon edema in coliticmice Our results imply thatdietary Gln prevented Th cell trafficking into colon tissuesand provide a new mechanism of Gln supplementation thathas beneficial effects on ameliorating the severity of acuteDSS-induced colitis

Conflict of Interests

The authors declare that they have no conflict of interests

12 Mediators of Inflammation

50120583m

CD3

50120583m

50120583m

50120583m

C

G

DC

DG

(a)

CD8

50120583m

50120583m

50120583m

50120583m

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 10 Representative immunofluorescence images of cytotoxic T cells In the left column cell surface antigens were stained for CD3(green FITC) and the staining of CD8 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI (blue)and the last column presents a colocalized fluorescence image of CD3+CD8+ T cells

Acknowledgments

The authors would like to thank Man-Hui Pai Assistant Pro-fessor of Department of Anatomy Taipei Medical Universityfor her technical assistance in immunofluorescence stainingThis studywas supported by aResearchGrant (NSC100-2320-B-038-007-MY3) from the National Science Council TaipeiTaiwan

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] C T Murphy K Nally F Shanahan and S Melgar ldquoShining alight on intestinal trafficrdquoClinical and Developmental Immunol-ogy vol 2012 Article ID 808157 14 pages 2012

Mediators of Inflammation 13

[3] L A Zenewicz A Antov and R A Flavell ldquoCD4 T-cell differ-entiation and inflammatory bowel diseaserdquo Trends inMolecularMedicine vol 15 no 5 pp 199ndash207 2009

[4] J C Lee P A Lyons E F McKinney et al ldquoGene expressionprofiling of CD8+ T cells predicts prognosis in patients withCrohn disease and ulcerative colitisrdquo Journal of Clinical Inves-tigation vol 121 no 10 pp 4170ndash4179 2011

[5] N T Funderburg S R Stubblefield Park H C Sung et alldquoCirculating CD4+ and CD8+ T cells are activated in IBDand are associated with plasma markers of inflammationrdquoImmunology vol 140 no 1 pp 87ndash97 2013

[6] E J Villablanca B Cassani U H von Andrian and J RMora ldquoBlocking lymphocyte localization to the gastrointestinalmucosa as a therapeutic strategy for inflammatory boweldiseasesrdquo Gastroenterology vol 140 no 6 pp 1776ndash1784 2011

[7] R P McEver and R D Cummings ldquoPerspectives series celladhesion in vascular biology Role of PSGL-1 binding toselectins in leukocyte recruitmentrdquo The Journal of ClinicalInvestigation vol 100 no 3 pp 485ndash491 1997

[8] C Berlin E L Berg M J Briskin et al ldquo12057241205737 Integrin medi-ates lymphocyte binding to the mucosal vascular addressinMAdCAM-1rdquo Cell vol 74 no 1 pp 185ndash195 1993

[9] K Ley C Laudanna M I Cybulsky and S Nourshargh ldquoGet-ting to the site of inflammation the leukocyte adhesion cascadeupdatedrdquoNature Reviews Immunology vol 7 no 9 pp 678ndash6892007

[10] B Moser M Wolf A Walz and P Loetscher ldquoChemokinesmultiple levels of leukocyte migration controlrdquo Trends inImmunology vol 25 no 2 pp 75ndash84 2004

[11] S Arimilli W Ferlin N Solvason S Deshpande M Howardand S Mocci ldquoChemokines in autoimmune diseasesrdquo Immuno-logical Reviews vol 177 pp 43ndash51 2000

[12] C Koenecke and R Forster ldquoCCR9 and inflammatory boweldiseaserdquo Expert Opinion on Therapeutic Targets vol 13 no 3pp 297ndash306 2009

[13] B Johansson-Lindbom and W W Agace ldquoGeneration of gut-homing T cells and their localization to the small intestinalmucosardquo Immunological Reviews vol 215 no 1 pp 226ndash2422007

[14] P EWischmeyer ldquoGlutaminemode of action in critical illnessrdquoCritical Care Medicine vol 35 no 9 pp S541ndashS544 2007

[15] N A Kretzmann H Fillmann J L Mauriz C A MarroniJ Gonzalez-Gallego and M J Tunon ldquoEffects of glutamineon proinflammatory gene expression and activation of nuclearfactor Kappa B and signal transducers and activators oftranscription in TNBS-induced colitisrdquo Inflammatory BowelDiseases vol 14 no 11 pp 1504ndash1513 2008

[16] H Xue A J D Sufit and P EWischmeyer ldquoGlutamine therapyimproves outcome of in vitro and in vivo experimental colitismodelsrdquo Journal of Parenteral and Enteral Nutrition vol 35 no2 pp 188ndash197 2011

[17] C-C Chu Y-C Hou M-H Pai C-J Chao and S-L YehldquoPretreatment with alanyl-glutamine suppresses T-helper-cell-associated cytokine expression and reduces inflammatoryresponses in mice with acute DSS-induced colitisrdquo The Journalof Nutritional Biochemistry vol 23 no 9 pp 1092ndash1099 2011

[18] S Kew S M Wells P Yaqoob F A Wallace E A Miles and PC Calder ldquoDietary glutamine enhances murine T-lymphocyteresponsivenessrdquo The Journal of Nutrition vol 129 no 8 pp1524ndash1531 1999

[19] S M Wells S Kew P Yaqoob F A Wallace and P C CalderldquoDietary glutamine enhances cytokine production by murinemacrophagesrdquo Nutrition vol 15 no 11-12 pp 881ndash884 1999

[20] Y-C Hou M-H Pai W-C Chiu Y-M Hu and S-L YehldquoEffects of dietary glutamine supplementation on lung injuryinduced by lipopolysaccharide administrationrdquo American Jour-nal of PhysiologymdashLung Cellular and Molecular Physiology vol296 no 3 pp L288ndashL295 2009

[21] M Kawada A Arihiro and E Mizoguchi ldquoInsights fromadvances in research of chemically induced experimental mod-els of human inflammatory bowel diseaserdquo World Journal ofGastroenterology vol 13 no 42 pp 5581ndash5593 2007

[22] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[23] R Yazbeck G S Howarth R N Butler M S Geier and CA Abbott ldquoBiochemical and histological changes in the smallintestine of mice with dextran sulfate sodium colitisrdquo Journal ofCellular Physiology vol 226 no 12 pp 3219ndash3224 2011

[24] M Mahler I J Bristol E H Leiter et al ldquoDifferentialsusceptibility of inbred mouse strains to dextran sulfatesodium-induced colitisrdquo American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 274 no 3 part 1 ppG544ndashG551 1998

[25] S Melgar A Karlsson and E Michaelsson ldquoAcute colitisinduced by dextran sulfate sodium progresses to chronicityin C57BL6 but not in BALBc mice correlation betweensymptoms and inflammationrdquoAmerican Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 288 no 6 pp G1328ndashG1338 2005

[26] MKoizumiN King R Lobb C Benjamin andDK PodolskyldquoExpression of vascular adhesion molecules in inflammatorybowel diseaserdquo Gastroenterology vol 103 no 3 pp 840ndash8471992

[27] L Peyrin-Biroulet E V Loftus Jr J-F Colombel and W JSandborn ldquoLong-term complications extraintestinal manifes-tations and mortality in adult Crohnrsquos disease in population-based cohortsrdquo Inflammatory Bowel Diseases vol 17 no 1 pp471ndash478 2011

[28] D H Adams and B Eksteen ldquoAberrant homing of mucosal Tcells and extra-intestinal manifestations of inflammatory boweldiseaserdquoNature Reviews Immunology vol 6 no 3 pp 244ndash2512006

[29] TW Spahn H Herbst P D Rennert et al ldquoInduction of colitisinmice deficient of Peyerrsquos patches andmesenteric lymph nodesis associated with increased disease severity and formation ofcolonic lymphoid patchesrdquo The American Journal of Pathologyvol 161 no 6 pp 2273ndash2282 2002

[30] S Nancey S Holvoet I Graber et al ldquoCD8+ Cytotoxic T CellsInduce Relapsing Colitis in Normal Micerdquo Gastroenterologyvol 131 no 2 pp 485ndash496 2006

[31] E C Butcher and L J Picker ldquoLymphocyte homing and home-ostasisrdquo Science vol 272 no 5258 pp 60ndash66 1996

[32] C Berlin R F Bargatze J J Campbell et al ldquo1205724 Integrinsmediate lymphocyte attachment and rolling under physiologicflowrdquo Cell vol 80 no 3 pp 413ndash422 1995

[33] K A Papadakis J Prehn V Nelson et al ldquoThe role of thymus-expressed chemokine and its receptor CCR9 on lymphocytesin the regional specialization of the mucosal immune systemrdquoJournal of Immunology vol 165 no 9 pp 5069ndash5076 2000

14 Mediators of Inflammation

[34] M-A Wurbel M G McIntire P Dwyer and E FiebigerldquoCCL25CCR9 interactions regulate large intestinal inflamma-tion in a murine model of acute colitisrdquo PLoS ONE vol 6 no 1Article ID e16442 2011

[35] CWang E K Hanly LWWheeler M Kaur K GMcDonaldand R D Newberry ldquoEffect of 12057241205737 blockade on intestinallymphocyte subsets and lymphoid tissue developmentrdquo Inflam-matory Bowel Diseases vol 16 no 10 pp 1751ndash1762 2010

[36] M Coeffier R Marion-Letellier and P Dechelotte ldquoPotentialfor amino acids supplementation during inflammatory boweldiseasesrdquo Inflammatory Bowel Diseases vol 16 no 3 pp 518ndash524 2010

[37] B San-Miguel I Crespo N A Kretzmann et al ldquoGlutamineprevents fibrosis development in rats with colitis induced by246-trinitrobenzene sulfonic acidrdquo The Journal of Nutritionvol 140 no 6 pp 1065ndash1071 2010

[38] Y C Hou C C Chu T L Ko et al ldquoEffects of alanyl-glutaminedipeptide on the expression of colon-inflammatory mediatorsduring the recovery phase of colitis induced by dextran sulfatesodiumrdquo European Journal of Nutrition vol 52 no 3 pp 1089ndash1098 2013

[39] Y Shiomi S Nishiumi M Ooi et al ldquoGCMS-based meta-bolomic study in mice with colitis induced by dextran sulfatesodiumrdquo Inflammatory Bowel Diseases vol 17 no 11 pp 2261ndash2274 2011

[40] M Vicario C Amat M Rivero M Moreto and C PelegrıldquoDietary glutamine affects mucosal functions in rats with mildDSS-induced colitisrdquoThe Journal of Nutrition vol 137 no 8 pp1931ndash1937 2007

[41] H Arndt F Kullmann F Reuszlig J Scholmerich and K-D Pal-itzsch ldquoGlutamine attenuates leukocyte-endothelial cell adhe-sion in indomethacin-induced intestinal inflammation in theratrdquo Journal of Parenteral and Enteral Nutrition vol 23 no 1pp 12ndash18 1999

[42] P Yaqoob and P C Calder ldquoGlutamine requirement of prolif-erating T lymphocytesrdquo Nutrition vol 13 no 7-8 pp 646ndash6511997

[43] E L Carr A Kelman G S Wu et al ldquoGlutamine uptake andmetabolism are coordinately regulated by ERKMAPK duringT lymphocyte activationrdquo Journal of Immunology vol 185 no2 pp 1037ndash1044 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Mediators of Inflammation

0

10

20

30

40

50

C G DC DG

PSG

L-1

()

lowast

lowast

dagger

(a)

0

2

4

6

8

10

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

5

10

15

20

25

C G DC DG

dagger

lowast

CD11

a (

)

(c)

0

10

20

30

40

50

C G DC DG

dagger

lowast

lowastCCR9

(

)

(d)

0

15

30

45

60

C G DC DG

lowast

CCR

9 ex

pres

sing

dagger

CD4+12057241205737+

T ce

lls (

)

(e)

Figure 4 Percentage of adhesionmolecule- and chemokine receptor-expressingThelper (Th) cells in blood CD4-positive blood lymphocyteswere gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d)) (e) Expression of CCR9 by CD4and 12057241205737 integrin double-positive blood lymphocytes Values are shown as the mean plusmn SEM lowastSignificantly different from the C group(119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

35 Gene Expression of Endothelial Adhesion Molecules inColon Tissues There was no difference in mRNA levels ofICAM-1 P-selectin and E-selectin between the C and Ggroups in colon tissues DSS-induced colitis greatly upreg-ulated the adhesion molecule genes expressed by activatedendothelial cells Compared to the DC group the expression

levels of ICAM-1 P-selectin and E-selectin mRNA weresuppressed in the DG group (Figure 8)

36 T Lymphocyte Subsets in the Colon Mucosa CD3 is thecell surface marker of T cells CD3 and CD4 double-positivecells are considered Th cells whereas Tc cells coexpress CD3

Mediators of Inflammation 7

0

10

20

30

40

50

C G DC DG

PSG

L-1

()

lowast

lowast

dagger

(a)

0

2

4

6

8

10

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

10

20

30

40

50

C G DC DG

CD11

a (

)

dagger

lowast

(c)

0

10

20

30

40

50

C G DC DG

dagger

lowast

CCR9

(

)

(d)

0

15

30

45

60

C G DC DG

dagger

lowast

CCR

9 ex

pres

sing

CD4+12057241205737+

T ce

lls (

)

(e)

Figure 5 Percentage of adhesion molecule- and chemokine receptor-expressing T helper (Th) cells in mesenteric lymph nodes (MLNs)CD4-positiveMLN lymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d))(e) Expression of CCR9 by CD4 and 12057241205737 integrin double-positive MLN lymphocytes Values are shown as the mean plusmn SEM lowastSignificantlydifferent from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

andCD8As shown in Figure 9 the immunoreactive intensityof Th cells was higher in the DC group than the DG groupHowever intensities of Tc cell populations did not differbetween the DC and DG groups (Figure 10)

4 Discussion

DSS is a heparin-like polysaccharide which results in acutechemical toxicity that disrupts the intestinal epithelial cell

8 Mediators of Inflammation

0

10

20

30

40

50

C G DC DG

lowast lowast

PSG

L-1

()

(a)

0

5

10

15

20

25

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

5

10

15

20

25

C G DC DG

dagger

lowast

CD11

a (

)

(c)

0

10

20

30

40

50

C G DC DG

CCR9

()

lowast

lowast

(d)

0

20

40

60

80

100

C G DC DG

lowastlowast

CCR

9 ex

pres

sing

CD8+12057241205737+

T ce

lls (

)

(e)

Figure 6 Percentage of adhesion molecule- and chemokine receptor-expressing cytotoxic T (Tc) cells in blood CD8-positive bloodlymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d)) (e) Expressionof CCR9 by CD8 and 12057241205737 integrin double-positive blood lymphocytes Values are shown as the mean plusmn SEM lowastSignificantly different fromthe C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

barrier [21] DSS-induced colitis is characterized by extensivecrypt and epithelial cell damage with ulceration tissueedema and infiltration of immune cells predominantly in thedistal colon that mimics the histological features of UC [22]Recent studies indicated that DSS-induced morphological

and biochemical damage also extends to the small intestines[23] Susceptibilities to DSS-induced colitis differ in variousinbred mouse strains [24] A single cycle of DSS exposureto the C57BL6 strain was found to develop acute colitiswhich later proceeds to chronic inflammation and T cell

Mediators of Inflammation 9

C G DC DG

PSG

L-1

()

0

10

20

30

40

50

lowastlowast

(a)

CD11

a (

)

C G DC DG0

5

10

15

20

25

CD11

a (

)

12057241205737

inte

grin

s (

)

(b)

C G DC DG0

5

10

15

20

25

CD11

a (

)

dagger

lowast

(c)

CCR9

()

C G DC DG0

5

10

15

20

25

lowast

lowast

(d)

CCR

9 ex

pres

sing

0

15

30

45

60

lowastlowast

CD+ 812057241205737+

T ce

lls (

)

C G DC DG

(e)

Figure 7 Percentage of adhesion molecule- and chemokine receptor-expressing cytotoxic T (Tc) cells in mesenteric lymph nodes (MLNs)CD8-positiveMLN lymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d))(e) Expression of CCR9 by CD8 and 12057241205737 integrin double-positive MLN lymphocytes Values are shown as the mean plusmn SEM lowastSignificantlydifferent from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

migration to the colon plays an important role in theprogression to chronicity [25] Regarding the high sensitivityto DSS C57BL6 mice were used in this study to analyze theconsequences of DSS exposure on adhesion molecules and

chemokine receptors involved in T cell trafficking to theintestines

IBD is associated with a massive influx of immune cellsinto the gut Previous studies indicated that increased local

10 Mediators of Inflammation

ICAM-1 P-selectin E-selectin

Relat

ive m

RNA

ratio

0

50

100

150

200

1000

1250

1500

CG

DCDG

daggerdagger

dagger

lowast

lowastlowast

lowast

lowast

lowast

Figure 8 Gene expression of endothelial adhesion molecules incolon tissues mRNA levels were analyzed by a real-time PCR TheC group was used as a calibrator and the data were presented as thefold change in gene expression relative to the calibrator Values areshown as themean plusmn SEM lowastSignificantly different from the C group(119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

secretion of proinflammatory cytokines by the inflamedcolon leads to upregulated expressions of vascular adhesionmolecules resulting in a sustained influx of inflammatorycells [26] Although inflammation predominantly occurs inthe GI tract IBD patients are likely to develop extraintestinalmanifestations [27] that may be attributed to aberrant acti-vation and homing of T cells [28] A recent study indicatedthat circulating CD4+ and CD8+ T cells are activated in bothCD and UC patients and these cells were correlated withleakage of microbial products from the impaired intestinalbarrier [5] Also the severity of DSS-induced colitis wascorrelatedwith the immune response ofMLNs [29] A similarphenomenon was also observed in this study Our resultsindicated that expressions of adhesion molecules and CCR9on Th cells and Tc cells significantly increased in both bloodand MLNs after DSS exposure These findings suggest thatboth local and systemic T cells are activated The role of Thcells in the pathogenesis of IBD has been widely studiedDysregulation ofTh cells can lead to immune cell infiltrationinto the intestinal mucosa and cause persistent inflammation[3 6] The pathogenic role of Tc cells in IBD has been lessinvestigated A previous study showed that antigen-specificTc cells caused relapsing colitis in normal mice due to thecytolytic function against the intestinal epithelium [30] Leeet al [4] reported a gene expression profile of circulatingCD8+ T cells that predicted a more aggressive disease coursefor IBD patients However the modulatory mechanism of Tccells in IBD is still under investigation

Naıve T cells constantly recirculate between the bloodand secondary lymphoid organs Once activated in secondarylymphoid organs they become effector T cells that expressadhesion molecules and chemokine receptors which controltheir extravasation into nonlymphoid tissue sites [31] T cell

trafficking to the gut and gut-associated lymphoid tissues(GALTs) requires 12057241205737 integrins The ligand MAdCAM-1is constitutively expressed by the mucosal endothelium inthe small intestine and colon [32] CCR9 is thought toparticipate in the specific localization of T cells to the smallintestines because the sole ligand for CCR9 C-C chemokineligand 25 (CCL25) is strongly expressed by the small intesti-nal epithelium [33] However Wurbel et al [34] revealedthat CCL25 expression increased during the recovery phaseafter acute DSS administration suggesting a regulatory roleof CCR9CCL25 interactions during colonic inflammationIn this study percentages of PSGL-1- LFA-1- and CCR9-expressing T cells were upregulated in acute DSS colitiswhereas expression levels of 12057241205737 integrins in colitic micedid not differ from those of normal mice It is possible thatT cell trafficking into the gut during acute DSS exposure isless dependent on 12057241205737 integrins In support of our findingsWang et al [35] reported that localization of T cells to theintestines was relatively unaffected by 12057241205737 blockade duringacute DSS-induced colitis

Gln is a critical fuel source for enterocytes and immunecells Gln supplementation attenuates gut injury by a com-plex mechanism which involves protecting the epithelialbarrier function reducing oxidative stress and modulat-ing inflammatory responses [36] The local and systemicimmunomodulatory effects of Gln have been discussed invarious experimental colitis models via different adminis-tration routes Studies using rodents with trinitrobenzenesulfonic acid-induced colitis indicated that Gln given bythe rectal route inhibits nuclear factor (NF)-120581B- and STAT-mediated inflammation in colon tissues [15] and further pre-vents colon fibrosis through downregulating gene pathwaysthat contribute to the accumulation of matrix proteins [37]We recently demonstrated that intraperitoneal pretreatmentwith alanyl-Gln a Gln-containing dipeptide widely used inparenteral nutrition suppresses cytokine expression in bloodTh cells reduces NF-120581B-mediated inflammatory responsesin the colon and upregulates expressions of genes whichpromote recovery of the colonic mucosa [17 38]

Because most exogenous Gln is absorbed in the proximalsmall intestine it might not reach the inflamed colon at asufficient concentration tomodulate inflammatory responses[36] However the enteral route of Gln administrationstill showed protective effects against DSS-induced damageOral Gln supplementation reduced the feces water contentenhanced expression of HSPs in the colonic mucosa andameliorated colon injury caused byDSS exposure [16 39 40]A previous study indicated that oral Gln attenuated leukocyteadhesion and emigration in a rodentmodel of indomethacin-induced ileitis [41] In this study we demonstrated thatoral Gln administration suppressed adhesion molecules andCCR9 expressed by T cells and downregulated the mRNAlevels of adhesion molecules expressed by endothelium incolon tissues The histological findings also support theresults that Gln administration suppressed the infiltrationof Th cells into the colon mucosa Gln consumption is animportant component of T cell activation [42 43] Differentsusceptibilities ofThandTc cells toGln supplementationmaybe explained by a higher cell population of Th cells which

Mediators of Inflammation 11

50120583m

50120583m

50120583m

50120583m

CD3

C

G

DC

DG

(a)

50120583m

50120583m

50120583m

50120583m

CD4

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 9 Representative examples of the immunofluorescence staining of T helper cells In the left column cell surface antigens were stainedfor CD3 (green FITC) and staining of CD4 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI(blue) and the last column presents a colocalized fluorescence image of CD3+CD4+ T cells

competes as a Gln source with Tc cells Further studies areneeded to investigate the molecular mechanisms involved ingene expressions of adhesion molecules and CCR9 regulatedby Gln

In conclusion this study showed for the first time thatpretreatment with oral Gln reduced adhesion molecule- andCCR9-expressing T cells induced by DSS exposure Theinhibitory abilities against adhesion molecule and CCR9expressions were more obvious inTh cells than Tc cells AlsoGln supplementation reduced gene expressions of endothe-lial adhesion molecules in colons prevented BW loss and

attenuated colon edema in coliticmice Our results imply thatdietary Gln prevented Th cell trafficking into colon tissuesand provide a new mechanism of Gln supplementation thathas beneficial effects on ameliorating the severity of acuteDSS-induced colitis

Conflict of Interests

The authors declare that they have no conflict of interests

12 Mediators of Inflammation

50120583m

CD3

50120583m

50120583m

50120583m

C

G

DC

DG

(a)

CD8

50120583m

50120583m

50120583m

50120583m

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 10 Representative immunofluorescence images of cytotoxic T cells In the left column cell surface antigens were stained for CD3(green FITC) and the staining of CD8 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI (blue)and the last column presents a colocalized fluorescence image of CD3+CD8+ T cells

Acknowledgments

The authors would like to thank Man-Hui Pai Assistant Pro-fessor of Department of Anatomy Taipei Medical Universityfor her technical assistance in immunofluorescence stainingThis studywas supported by aResearchGrant (NSC100-2320-B-038-007-MY3) from the National Science Council TaipeiTaiwan

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] C T Murphy K Nally F Shanahan and S Melgar ldquoShining alight on intestinal trafficrdquoClinical and Developmental Immunol-ogy vol 2012 Article ID 808157 14 pages 2012

Mediators of Inflammation 13

[3] L A Zenewicz A Antov and R A Flavell ldquoCD4 T-cell differ-entiation and inflammatory bowel diseaserdquo Trends inMolecularMedicine vol 15 no 5 pp 199ndash207 2009

[4] J C Lee P A Lyons E F McKinney et al ldquoGene expressionprofiling of CD8+ T cells predicts prognosis in patients withCrohn disease and ulcerative colitisrdquo Journal of Clinical Inves-tigation vol 121 no 10 pp 4170ndash4179 2011

[5] N T Funderburg S R Stubblefield Park H C Sung et alldquoCirculating CD4+ and CD8+ T cells are activated in IBDand are associated with plasma markers of inflammationrdquoImmunology vol 140 no 1 pp 87ndash97 2013

[6] E J Villablanca B Cassani U H von Andrian and J RMora ldquoBlocking lymphocyte localization to the gastrointestinalmucosa as a therapeutic strategy for inflammatory boweldiseasesrdquo Gastroenterology vol 140 no 6 pp 1776ndash1784 2011

[7] R P McEver and R D Cummings ldquoPerspectives series celladhesion in vascular biology Role of PSGL-1 binding toselectins in leukocyte recruitmentrdquo The Journal of ClinicalInvestigation vol 100 no 3 pp 485ndash491 1997

[8] C Berlin E L Berg M J Briskin et al ldquo12057241205737 Integrin medi-ates lymphocyte binding to the mucosal vascular addressinMAdCAM-1rdquo Cell vol 74 no 1 pp 185ndash195 1993

[9] K Ley C Laudanna M I Cybulsky and S Nourshargh ldquoGet-ting to the site of inflammation the leukocyte adhesion cascadeupdatedrdquoNature Reviews Immunology vol 7 no 9 pp 678ndash6892007

[10] B Moser M Wolf A Walz and P Loetscher ldquoChemokinesmultiple levels of leukocyte migration controlrdquo Trends inImmunology vol 25 no 2 pp 75ndash84 2004

[11] S Arimilli W Ferlin N Solvason S Deshpande M Howardand S Mocci ldquoChemokines in autoimmune diseasesrdquo Immuno-logical Reviews vol 177 pp 43ndash51 2000

[12] C Koenecke and R Forster ldquoCCR9 and inflammatory boweldiseaserdquo Expert Opinion on Therapeutic Targets vol 13 no 3pp 297ndash306 2009

[13] B Johansson-Lindbom and W W Agace ldquoGeneration of gut-homing T cells and their localization to the small intestinalmucosardquo Immunological Reviews vol 215 no 1 pp 226ndash2422007

[14] P EWischmeyer ldquoGlutaminemode of action in critical illnessrdquoCritical Care Medicine vol 35 no 9 pp S541ndashS544 2007

[15] N A Kretzmann H Fillmann J L Mauriz C A MarroniJ Gonzalez-Gallego and M J Tunon ldquoEffects of glutamineon proinflammatory gene expression and activation of nuclearfactor Kappa B and signal transducers and activators oftranscription in TNBS-induced colitisrdquo Inflammatory BowelDiseases vol 14 no 11 pp 1504ndash1513 2008

[16] H Xue A J D Sufit and P EWischmeyer ldquoGlutamine therapyimproves outcome of in vitro and in vivo experimental colitismodelsrdquo Journal of Parenteral and Enteral Nutrition vol 35 no2 pp 188ndash197 2011

[17] C-C Chu Y-C Hou M-H Pai C-J Chao and S-L YehldquoPretreatment with alanyl-glutamine suppresses T-helper-cell-associated cytokine expression and reduces inflammatoryresponses in mice with acute DSS-induced colitisrdquo The Journalof Nutritional Biochemistry vol 23 no 9 pp 1092ndash1099 2011

[18] S Kew S M Wells P Yaqoob F A Wallace E A Miles and PC Calder ldquoDietary glutamine enhances murine T-lymphocyteresponsivenessrdquo The Journal of Nutrition vol 129 no 8 pp1524ndash1531 1999

[19] S M Wells S Kew P Yaqoob F A Wallace and P C CalderldquoDietary glutamine enhances cytokine production by murinemacrophagesrdquo Nutrition vol 15 no 11-12 pp 881ndash884 1999

[20] Y-C Hou M-H Pai W-C Chiu Y-M Hu and S-L YehldquoEffects of dietary glutamine supplementation on lung injuryinduced by lipopolysaccharide administrationrdquo American Jour-nal of PhysiologymdashLung Cellular and Molecular Physiology vol296 no 3 pp L288ndashL295 2009

[21] M Kawada A Arihiro and E Mizoguchi ldquoInsights fromadvances in research of chemically induced experimental mod-els of human inflammatory bowel diseaserdquo World Journal ofGastroenterology vol 13 no 42 pp 5581ndash5593 2007

[22] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[23] R Yazbeck G S Howarth R N Butler M S Geier and CA Abbott ldquoBiochemical and histological changes in the smallintestine of mice with dextran sulfate sodium colitisrdquo Journal ofCellular Physiology vol 226 no 12 pp 3219ndash3224 2011

[24] M Mahler I J Bristol E H Leiter et al ldquoDifferentialsusceptibility of inbred mouse strains to dextran sulfatesodium-induced colitisrdquo American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 274 no 3 part 1 ppG544ndashG551 1998

[25] S Melgar A Karlsson and E Michaelsson ldquoAcute colitisinduced by dextran sulfate sodium progresses to chronicityin C57BL6 but not in BALBc mice correlation betweensymptoms and inflammationrdquoAmerican Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 288 no 6 pp G1328ndashG1338 2005

[26] MKoizumiN King R Lobb C Benjamin andDK PodolskyldquoExpression of vascular adhesion molecules in inflammatorybowel diseaserdquo Gastroenterology vol 103 no 3 pp 840ndash8471992

[27] L Peyrin-Biroulet E V Loftus Jr J-F Colombel and W JSandborn ldquoLong-term complications extraintestinal manifes-tations and mortality in adult Crohnrsquos disease in population-based cohortsrdquo Inflammatory Bowel Diseases vol 17 no 1 pp471ndash478 2011

[28] D H Adams and B Eksteen ldquoAberrant homing of mucosal Tcells and extra-intestinal manifestations of inflammatory boweldiseaserdquoNature Reviews Immunology vol 6 no 3 pp 244ndash2512006

[29] TW Spahn H Herbst P D Rennert et al ldquoInduction of colitisinmice deficient of Peyerrsquos patches andmesenteric lymph nodesis associated with increased disease severity and formation ofcolonic lymphoid patchesrdquo The American Journal of Pathologyvol 161 no 6 pp 2273ndash2282 2002

[30] S Nancey S Holvoet I Graber et al ldquoCD8+ Cytotoxic T CellsInduce Relapsing Colitis in Normal Micerdquo Gastroenterologyvol 131 no 2 pp 485ndash496 2006

[31] E C Butcher and L J Picker ldquoLymphocyte homing and home-ostasisrdquo Science vol 272 no 5258 pp 60ndash66 1996

[32] C Berlin R F Bargatze J J Campbell et al ldquo1205724 Integrinsmediate lymphocyte attachment and rolling under physiologicflowrdquo Cell vol 80 no 3 pp 413ndash422 1995

[33] K A Papadakis J Prehn V Nelson et al ldquoThe role of thymus-expressed chemokine and its receptor CCR9 on lymphocytesin the regional specialization of the mucosal immune systemrdquoJournal of Immunology vol 165 no 9 pp 5069ndash5076 2000

14 Mediators of Inflammation

[34] M-A Wurbel M G McIntire P Dwyer and E FiebigerldquoCCL25CCR9 interactions regulate large intestinal inflamma-tion in a murine model of acute colitisrdquo PLoS ONE vol 6 no 1Article ID e16442 2011

[35] CWang E K Hanly LWWheeler M Kaur K GMcDonaldand R D Newberry ldquoEffect of 12057241205737 blockade on intestinallymphocyte subsets and lymphoid tissue developmentrdquo Inflam-matory Bowel Diseases vol 16 no 10 pp 1751ndash1762 2010

[36] M Coeffier R Marion-Letellier and P Dechelotte ldquoPotentialfor amino acids supplementation during inflammatory boweldiseasesrdquo Inflammatory Bowel Diseases vol 16 no 3 pp 518ndash524 2010

[37] B San-Miguel I Crespo N A Kretzmann et al ldquoGlutamineprevents fibrosis development in rats with colitis induced by246-trinitrobenzene sulfonic acidrdquo The Journal of Nutritionvol 140 no 6 pp 1065ndash1071 2010

[38] Y C Hou C C Chu T L Ko et al ldquoEffects of alanyl-glutaminedipeptide on the expression of colon-inflammatory mediatorsduring the recovery phase of colitis induced by dextran sulfatesodiumrdquo European Journal of Nutrition vol 52 no 3 pp 1089ndash1098 2013

[39] Y Shiomi S Nishiumi M Ooi et al ldquoGCMS-based meta-bolomic study in mice with colitis induced by dextran sulfatesodiumrdquo Inflammatory Bowel Diseases vol 17 no 11 pp 2261ndash2274 2011

[40] M Vicario C Amat M Rivero M Moreto and C PelegrıldquoDietary glutamine affects mucosal functions in rats with mildDSS-induced colitisrdquoThe Journal of Nutrition vol 137 no 8 pp1931ndash1937 2007

[41] H Arndt F Kullmann F Reuszlig J Scholmerich and K-D Pal-itzsch ldquoGlutamine attenuates leukocyte-endothelial cell adhe-sion in indomethacin-induced intestinal inflammation in theratrdquo Journal of Parenteral and Enteral Nutrition vol 23 no 1pp 12ndash18 1999

[42] P Yaqoob and P C Calder ldquoGlutamine requirement of prolif-erating T lymphocytesrdquo Nutrition vol 13 no 7-8 pp 646ndash6511997

[43] E L Carr A Kelman G S Wu et al ldquoGlutamine uptake andmetabolism are coordinately regulated by ERKMAPK duringT lymphocyte activationrdquo Journal of Immunology vol 185 no2 pp 1037ndash1044 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 7

0

10

20

30

40

50

C G DC DG

PSG

L-1

()

lowast

lowast

dagger

(a)

0

2

4

6

8

10

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

10

20

30

40

50

C G DC DG

CD11

a (

)

dagger

lowast

(c)

0

10

20

30

40

50

C G DC DG

dagger

lowast

CCR9

(

)

(d)

0

15

30

45

60

C G DC DG

dagger

lowast

CCR

9 ex

pres

sing

CD4+12057241205737+

T ce

lls (

)

(e)

Figure 5 Percentage of adhesion molecule- and chemokine receptor-expressing T helper (Th) cells in mesenteric lymph nodes (MLNs)CD4-positiveMLN lymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d))(e) Expression of CCR9 by CD4 and 12057241205737 integrin double-positive MLN lymphocytes Values are shown as the mean plusmn SEM lowastSignificantlydifferent from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

andCD8As shown in Figure 9 the immunoreactive intensityof Th cells was higher in the DC group than the DG groupHowever intensities of Tc cell populations did not differbetween the DC and DG groups (Figure 10)

4 Discussion

DSS is a heparin-like polysaccharide which results in acutechemical toxicity that disrupts the intestinal epithelial cell

8 Mediators of Inflammation

0

10

20

30

40

50

C G DC DG

lowast lowast

PSG

L-1

()

(a)

0

5

10

15

20

25

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

5

10

15

20

25

C G DC DG

dagger

lowast

CD11

a (

)

(c)

0

10

20

30

40

50

C G DC DG

CCR9

()

lowast

lowast

(d)

0

20

40

60

80

100

C G DC DG

lowastlowast

CCR

9 ex

pres

sing

CD8+12057241205737+

T ce

lls (

)

(e)

Figure 6 Percentage of adhesion molecule- and chemokine receptor-expressing cytotoxic T (Tc) cells in blood CD8-positive bloodlymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d)) (e) Expressionof CCR9 by CD8 and 12057241205737 integrin double-positive blood lymphocytes Values are shown as the mean plusmn SEM lowastSignificantly different fromthe C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

barrier [21] DSS-induced colitis is characterized by extensivecrypt and epithelial cell damage with ulceration tissueedema and infiltration of immune cells predominantly in thedistal colon that mimics the histological features of UC [22]Recent studies indicated that DSS-induced morphological

and biochemical damage also extends to the small intestines[23] Susceptibilities to DSS-induced colitis differ in variousinbred mouse strains [24] A single cycle of DSS exposureto the C57BL6 strain was found to develop acute colitiswhich later proceeds to chronic inflammation and T cell

Mediators of Inflammation 9

C G DC DG

PSG

L-1

()

0

10

20

30

40

50

lowastlowast

(a)

CD11

a (

)

C G DC DG0

5

10

15

20

25

CD11

a (

)

12057241205737

inte

grin

s (

)

(b)

C G DC DG0

5

10

15

20

25

CD11

a (

)

dagger

lowast

(c)

CCR9

()

C G DC DG0

5

10

15

20

25

lowast

lowast

(d)

CCR

9 ex

pres

sing

0

15

30

45

60

lowastlowast

CD+ 812057241205737+

T ce

lls (

)

C G DC DG

(e)

Figure 7 Percentage of adhesion molecule- and chemokine receptor-expressing cytotoxic T (Tc) cells in mesenteric lymph nodes (MLNs)CD8-positiveMLN lymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d))(e) Expression of CCR9 by CD8 and 12057241205737 integrin double-positive MLN lymphocytes Values are shown as the mean plusmn SEM lowastSignificantlydifferent from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

migration to the colon plays an important role in theprogression to chronicity [25] Regarding the high sensitivityto DSS C57BL6 mice were used in this study to analyze theconsequences of DSS exposure on adhesion molecules and

chemokine receptors involved in T cell trafficking to theintestines

IBD is associated with a massive influx of immune cellsinto the gut Previous studies indicated that increased local

10 Mediators of Inflammation

ICAM-1 P-selectin E-selectin

Relat

ive m

RNA

ratio

0

50

100

150

200

1000

1250

1500

CG

DCDG

daggerdagger

dagger

lowast

lowastlowast

lowast

lowast

lowast

Figure 8 Gene expression of endothelial adhesion molecules incolon tissues mRNA levels were analyzed by a real-time PCR TheC group was used as a calibrator and the data were presented as thefold change in gene expression relative to the calibrator Values areshown as themean plusmn SEM lowastSignificantly different from the C group(119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

secretion of proinflammatory cytokines by the inflamedcolon leads to upregulated expressions of vascular adhesionmolecules resulting in a sustained influx of inflammatorycells [26] Although inflammation predominantly occurs inthe GI tract IBD patients are likely to develop extraintestinalmanifestations [27] that may be attributed to aberrant acti-vation and homing of T cells [28] A recent study indicatedthat circulating CD4+ and CD8+ T cells are activated in bothCD and UC patients and these cells were correlated withleakage of microbial products from the impaired intestinalbarrier [5] Also the severity of DSS-induced colitis wascorrelatedwith the immune response ofMLNs [29] A similarphenomenon was also observed in this study Our resultsindicated that expressions of adhesion molecules and CCR9on Th cells and Tc cells significantly increased in both bloodand MLNs after DSS exposure These findings suggest thatboth local and systemic T cells are activated The role of Thcells in the pathogenesis of IBD has been widely studiedDysregulation ofTh cells can lead to immune cell infiltrationinto the intestinal mucosa and cause persistent inflammation[3 6] The pathogenic role of Tc cells in IBD has been lessinvestigated A previous study showed that antigen-specificTc cells caused relapsing colitis in normal mice due to thecytolytic function against the intestinal epithelium [30] Leeet al [4] reported a gene expression profile of circulatingCD8+ T cells that predicted a more aggressive disease coursefor IBD patients However the modulatory mechanism of Tccells in IBD is still under investigation

Naıve T cells constantly recirculate between the bloodand secondary lymphoid organs Once activated in secondarylymphoid organs they become effector T cells that expressadhesion molecules and chemokine receptors which controltheir extravasation into nonlymphoid tissue sites [31] T cell

trafficking to the gut and gut-associated lymphoid tissues(GALTs) requires 12057241205737 integrins The ligand MAdCAM-1is constitutively expressed by the mucosal endothelium inthe small intestine and colon [32] CCR9 is thought toparticipate in the specific localization of T cells to the smallintestines because the sole ligand for CCR9 C-C chemokineligand 25 (CCL25) is strongly expressed by the small intesti-nal epithelium [33] However Wurbel et al [34] revealedthat CCL25 expression increased during the recovery phaseafter acute DSS administration suggesting a regulatory roleof CCR9CCL25 interactions during colonic inflammationIn this study percentages of PSGL-1- LFA-1- and CCR9-expressing T cells were upregulated in acute DSS colitiswhereas expression levels of 12057241205737 integrins in colitic micedid not differ from those of normal mice It is possible thatT cell trafficking into the gut during acute DSS exposure isless dependent on 12057241205737 integrins In support of our findingsWang et al [35] reported that localization of T cells to theintestines was relatively unaffected by 12057241205737 blockade duringacute DSS-induced colitis

Gln is a critical fuel source for enterocytes and immunecells Gln supplementation attenuates gut injury by a com-plex mechanism which involves protecting the epithelialbarrier function reducing oxidative stress and modulat-ing inflammatory responses [36] The local and systemicimmunomodulatory effects of Gln have been discussed invarious experimental colitis models via different adminis-tration routes Studies using rodents with trinitrobenzenesulfonic acid-induced colitis indicated that Gln given bythe rectal route inhibits nuclear factor (NF)-120581B- and STAT-mediated inflammation in colon tissues [15] and further pre-vents colon fibrosis through downregulating gene pathwaysthat contribute to the accumulation of matrix proteins [37]We recently demonstrated that intraperitoneal pretreatmentwith alanyl-Gln a Gln-containing dipeptide widely used inparenteral nutrition suppresses cytokine expression in bloodTh cells reduces NF-120581B-mediated inflammatory responsesin the colon and upregulates expressions of genes whichpromote recovery of the colonic mucosa [17 38]

Because most exogenous Gln is absorbed in the proximalsmall intestine it might not reach the inflamed colon at asufficient concentration tomodulate inflammatory responses[36] However the enteral route of Gln administrationstill showed protective effects against DSS-induced damageOral Gln supplementation reduced the feces water contentenhanced expression of HSPs in the colonic mucosa andameliorated colon injury caused byDSS exposure [16 39 40]A previous study indicated that oral Gln attenuated leukocyteadhesion and emigration in a rodentmodel of indomethacin-induced ileitis [41] In this study we demonstrated thatoral Gln administration suppressed adhesion molecules andCCR9 expressed by T cells and downregulated the mRNAlevels of adhesion molecules expressed by endothelium incolon tissues The histological findings also support theresults that Gln administration suppressed the infiltrationof Th cells into the colon mucosa Gln consumption is animportant component of T cell activation [42 43] Differentsusceptibilities ofThandTc cells toGln supplementationmaybe explained by a higher cell population of Th cells which

Mediators of Inflammation 11

50120583m

50120583m

50120583m

50120583m

CD3

C

G

DC

DG

(a)

50120583m

50120583m

50120583m

50120583m

CD4

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 9 Representative examples of the immunofluorescence staining of T helper cells In the left column cell surface antigens were stainedfor CD3 (green FITC) and staining of CD4 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI(blue) and the last column presents a colocalized fluorescence image of CD3+CD4+ T cells

competes as a Gln source with Tc cells Further studies areneeded to investigate the molecular mechanisms involved ingene expressions of adhesion molecules and CCR9 regulatedby Gln

In conclusion this study showed for the first time thatpretreatment with oral Gln reduced adhesion molecule- andCCR9-expressing T cells induced by DSS exposure Theinhibitory abilities against adhesion molecule and CCR9expressions were more obvious inTh cells than Tc cells AlsoGln supplementation reduced gene expressions of endothe-lial adhesion molecules in colons prevented BW loss and

attenuated colon edema in coliticmice Our results imply thatdietary Gln prevented Th cell trafficking into colon tissuesand provide a new mechanism of Gln supplementation thathas beneficial effects on ameliorating the severity of acuteDSS-induced colitis

Conflict of Interests

The authors declare that they have no conflict of interests

12 Mediators of Inflammation

50120583m

CD3

50120583m

50120583m

50120583m

C

G

DC

DG

(a)

CD8

50120583m

50120583m

50120583m

50120583m

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 10 Representative immunofluorescence images of cytotoxic T cells In the left column cell surface antigens were stained for CD3(green FITC) and the staining of CD8 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI (blue)and the last column presents a colocalized fluorescence image of CD3+CD8+ T cells

Acknowledgments

The authors would like to thank Man-Hui Pai Assistant Pro-fessor of Department of Anatomy Taipei Medical Universityfor her technical assistance in immunofluorescence stainingThis studywas supported by aResearchGrant (NSC100-2320-B-038-007-MY3) from the National Science Council TaipeiTaiwan

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] C T Murphy K Nally F Shanahan and S Melgar ldquoShining alight on intestinal trafficrdquoClinical and Developmental Immunol-ogy vol 2012 Article ID 808157 14 pages 2012

Mediators of Inflammation 13

[3] L A Zenewicz A Antov and R A Flavell ldquoCD4 T-cell differ-entiation and inflammatory bowel diseaserdquo Trends inMolecularMedicine vol 15 no 5 pp 199ndash207 2009

[4] J C Lee P A Lyons E F McKinney et al ldquoGene expressionprofiling of CD8+ T cells predicts prognosis in patients withCrohn disease and ulcerative colitisrdquo Journal of Clinical Inves-tigation vol 121 no 10 pp 4170ndash4179 2011

[5] N T Funderburg S R Stubblefield Park H C Sung et alldquoCirculating CD4+ and CD8+ T cells are activated in IBDand are associated with plasma markers of inflammationrdquoImmunology vol 140 no 1 pp 87ndash97 2013

[6] E J Villablanca B Cassani U H von Andrian and J RMora ldquoBlocking lymphocyte localization to the gastrointestinalmucosa as a therapeutic strategy for inflammatory boweldiseasesrdquo Gastroenterology vol 140 no 6 pp 1776ndash1784 2011

[7] R P McEver and R D Cummings ldquoPerspectives series celladhesion in vascular biology Role of PSGL-1 binding toselectins in leukocyte recruitmentrdquo The Journal of ClinicalInvestigation vol 100 no 3 pp 485ndash491 1997

[8] C Berlin E L Berg M J Briskin et al ldquo12057241205737 Integrin medi-ates lymphocyte binding to the mucosal vascular addressinMAdCAM-1rdquo Cell vol 74 no 1 pp 185ndash195 1993

[9] K Ley C Laudanna M I Cybulsky and S Nourshargh ldquoGet-ting to the site of inflammation the leukocyte adhesion cascadeupdatedrdquoNature Reviews Immunology vol 7 no 9 pp 678ndash6892007

[10] B Moser M Wolf A Walz and P Loetscher ldquoChemokinesmultiple levels of leukocyte migration controlrdquo Trends inImmunology vol 25 no 2 pp 75ndash84 2004

[11] S Arimilli W Ferlin N Solvason S Deshpande M Howardand S Mocci ldquoChemokines in autoimmune diseasesrdquo Immuno-logical Reviews vol 177 pp 43ndash51 2000

[12] C Koenecke and R Forster ldquoCCR9 and inflammatory boweldiseaserdquo Expert Opinion on Therapeutic Targets vol 13 no 3pp 297ndash306 2009

[13] B Johansson-Lindbom and W W Agace ldquoGeneration of gut-homing T cells and their localization to the small intestinalmucosardquo Immunological Reviews vol 215 no 1 pp 226ndash2422007

[14] P EWischmeyer ldquoGlutaminemode of action in critical illnessrdquoCritical Care Medicine vol 35 no 9 pp S541ndashS544 2007

[15] N A Kretzmann H Fillmann J L Mauriz C A MarroniJ Gonzalez-Gallego and M J Tunon ldquoEffects of glutamineon proinflammatory gene expression and activation of nuclearfactor Kappa B and signal transducers and activators oftranscription in TNBS-induced colitisrdquo Inflammatory BowelDiseases vol 14 no 11 pp 1504ndash1513 2008

[16] H Xue A J D Sufit and P EWischmeyer ldquoGlutamine therapyimproves outcome of in vitro and in vivo experimental colitismodelsrdquo Journal of Parenteral and Enteral Nutrition vol 35 no2 pp 188ndash197 2011

[17] C-C Chu Y-C Hou M-H Pai C-J Chao and S-L YehldquoPretreatment with alanyl-glutamine suppresses T-helper-cell-associated cytokine expression and reduces inflammatoryresponses in mice with acute DSS-induced colitisrdquo The Journalof Nutritional Biochemistry vol 23 no 9 pp 1092ndash1099 2011

[18] S Kew S M Wells P Yaqoob F A Wallace E A Miles and PC Calder ldquoDietary glutamine enhances murine T-lymphocyteresponsivenessrdquo The Journal of Nutrition vol 129 no 8 pp1524ndash1531 1999

[19] S M Wells S Kew P Yaqoob F A Wallace and P C CalderldquoDietary glutamine enhances cytokine production by murinemacrophagesrdquo Nutrition vol 15 no 11-12 pp 881ndash884 1999

[20] Y-C Hou M-H Pai W-C Chiu Y-M Hu and S-L YehldquoEffects of dietary glutamine supplementation on lung injuryinduced by lipopolysaccharide administrationrdquo American Jour-nal of PhysiologymdashLung Cellular and Molecular Physiology vol296 no 3 pp L288ndashL295 2009

[21] M Kawada A Arihiro and E Mizoguchi ldquoInsights fromadvances in research of chemically induced experimental mod-els of human inflammatory bowel diseaserdquo World Journal ofGastroenterology vol 13 no 42 pp 5581ndash5593 2007

[22] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[23] R Yazbeck G S Howarth R N Butler M S Geier and CA Abbott ldquoBiochemical and histological changes in the smallintestine of mice with dextran sulfate sodium colitisrdquo Journal ofCellular Physiology vol 226 no 12 pp 3219ndash3224 2011

[24] M Mahler I J Bristol E H Leiter et al ldquoDifferentialsusceptibility of inbred mouse strains to dextran sulfatesodium-induced colitisrdquo American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 274 no 3 part 1 ppG544ndashG551 1998

[25] S Melgar A Karlsson and E Michaelsson ldquoAcute colitisinduced by dextran sulfate sodium progresses to chronicityin C57BL6 but not in BALBc mice correlation betweensymptoms and inflammationrdquoAmerican Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 288 no 6 pp G1328ndashG1338 2005

[26] MKoizumiN King R Lobb C Benjamin andDK PodolskyldquoExpression of vascular adhesion molecules in inflammatorybowel diseaserdquo Gastroenterology vol 103 no 3 pp 840ndash8471992

[27] L Peyrin-Biroulet E V Loftus Jr J-F Colombel and W JSandborn ldquoLong-term complications extraintestinal manifes-tations and mortality in adult Crohnrsquos disease in population-based cohortsrdquo Inflammatory Bowel Diseases vol 17 no 1 pp471ndash478 2011

[28] D H Adams and B Eksteen ldquoAberrant homing of mucosal Tcells and extra-intestinal manifestations of inflammatory boweldiseaserdquoNature Reviews Immunology vol 6 no 3 pp 244ndash2512006

[29] TW Spahn H Herbst P D Rennert et al ldquoInduction of colitisinmice deficient of Peyerrsquos patches andmesenteric lymph nodesis associated with increased disease severity and formation ofcolonic lymphoid patchesrdquo The American Journal of Pathologyvol 161 no 6 pp 2273ndash2282 2002

[30] S Nancey S Holvoet I Graber et al ldquoCD8+ Cytotoxic T CellsInduce Relapsing Colitis in Normal Micerdquo Gastroenterologyvol 131 no 2 pp 485ndash496 2006

[31] E C Butcher and L J Picker ldquoLymphocyte homing and home-ostasisrdquo Science vol 272 no 5258 pp 60ndash66 1996

[32] C Berlin R F Bargatze J J Campbell et al ldquo1205724 Integrinsmediate lymphocyte attachment and rolling under physiologicflowrdquo Cell vol 80 no 3 pp 413ndash422 1995

[33] K A Papadakis J Prehn V Nelson et al ldquoThe role of thymus-expressed chemokine and its receptor CCR9 on lymphocytesin the regional specialization of the mucosal immune systemrdquoJournal of Immunology vol 165 no 9 pp 5069ndash5076 2000

14 Mediators of Inflammation

[34] M-A Wurbel M G McIntire P Dwyer and E FiebigerldquoCCL25CCR9 interactions regulate large intestinal inflamma-tion in a murine model of acute colitisrdquo PLoS ONE vol 6 no 1Article ID e16442 2011

[35] CWang E K Hanly LWWheeler M Kaur K GMcDonaldand R D Newberry ldquoEffect of 12057241205737 blockade on intestinallymphocyte subsets and lymphoid tissue developmentrdquo Inflam-matory Bowel Diseases vol 16 no 10 pp 1751ndash1762 2010

[36] M Coeffier R Marion-Letellier and P Dechelotte ldquoPotentialfor amino acids supplementation during inflammatory boweldiseasesrdquo Inflammatory Bowel Diseases vol 16 no 3 pp 518ndash524 2010

[37] B San-Miguel I Crespo N A Kretzmann et al ldquoGlutamineprevents fibrosis development in rats with colitis induced by246-trinitrobenzene sulfonic acidrdquo The Journal of Nutritionvol 140 no 6 pp 1065ndash1071 2010

[38] Y C Hou C C Chu T L Ko et al ldquoEffects of alanyl-glutaminedipeptide on the expression of colon-inflammatory mediatorsduring the recovery phase of colitis induced by dextran sulfatesodiumrdquo European Journal of Nutrition vol 52 no 3 pp 1089ndash1098 2013

[39] Y Shiomi S Nishiumi M Ooi et al ldquoGCMS-based meta-bolomic study in mice with colitis induced by dextran sulfatesodiumrdquo Inflammatory Bowel Diseases vol 17 no 11 pp 2261ndash2274 2011

[40] M Vicario C Amat M Rivero M Moreto and C PelegrıldquoDietary glutamine affects mucosal functions in rats with mildDSS-induced colitisrdquoThe Journal of Nutrition vol 137 no 8 pp1931ndash1937 2007

[41] H Arndt F Kullmann F Reuszlig J Scholmerich and K-D Pal-itzsch ldquoGlutamine attenuates leukocyte-endothelial cell adhe-sion in indomethacin-induced intestinal inflammation in theratrdquo Journal of Parenteral and Enteral Nutrition vol 23 no 1pp 12ndash18 1999

[42] P Yaqoob and P C Calder ldquoGlutamine requirement of prolif-erating T lymphocytesrdquo Nutrition vol 13 no 7-8 pp 646ndash6511997

[43] E L Carr A Kelman G S Wu et al ldquoGlutamine uptake andmetabolism are coordinately regulated by ERKMAPK duringT lymphocyte activationrdquo Journal of Immunology vol 185 no2 pp 1037ndash1044 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Mediators of Inflammation

0

10

20

30

40

50

C G DC DG

lowast lowast

PSG

L-1

()

(a)

0

5

10

15

20

25

C G DC DG

12057241205737

inte

grin

s (

)

(b)

0

5

10

15

20

25

C G DC DG

dagger

lowast

CD11

a (

)

(c)

0

10

20

30

40

50

C G DC DG

CCR9

()

lowast

lowast

(d)

0

20

40

60

80

100

C G DC DG

lowastlowast

CCR

9 ex

pres

sing

CD8+12057241205737+

T ce

lls (

)

(e)

Figure 6 Percentage of adhesion molecule- and chemokine receptor-expressing cytotoxic T (Tc) cells in blood CD8-positive bloodlymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d)) (e) Expressionof CCR9 by CD8 and 12057241205737 integrin double-positive blood lymphocytes Values are shown as the mean plusmn SEM lowastSignificantly different fromthe C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

barrier [21] DSS-induced colitis is characterized by extensivecrypt and epithelial cell damage with ulceration tissueedema and infiltration of immune cells predominantly in thedistal colon that mimics the histological features of UC [22]Recent studies indicated that DSS-induced morphological

and biochemical damage also extends to the small intestines[23] Susceptibilities to DSS-induced colitis differ in variousinbred mouse strains [24] A single cycle of DSS exposureto the C57BL6 strain was found to develop acute colitiswhich later proceeds to chronic inflammation and T cell

Mediators of Inflammation 9

C G DC DG

PSG

L-1

()

0

10

20

30

40

50

lowastlowast

(a)

CD11

a (

)

C G DC DG0

5

10

15

20

25

CD11

a (

)

12057241205737

inte

grin

s (

)

(b)

C G DC DG0

5

10

15

20

25

CD11

a (

)

dagger

lowast

(c)

CCR9

()

C G DC DG0

5

10

15

20

25

lowast

lowast

(d)

CCR

9 ex

pres

sing

0

15

30

45

60

lowastlowast

CD+ 812057241205737+

T ce

lls (

)

C G DC DG

(e)

Figure 7 Percentage of adhesion molecule- and chemokine receptor-expressing cytotoxic T (Tc) cells in mesenteric lymph nodes (MLNs)CD8-positiveMLN lymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d))(e) Expression of CCR9 by CD8 and 12057241205737 integrin double-positive MLN lymphocytes Values are shown as the mean plusmn SEM lowastSignificantlydifferent from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

migration to the colon plays an important role in theprogression to chronicity [25] Regarding the high sensitivityto DSS C57BL6 mice were used in this study to analyze theconsequences of DSS exposure on adhesion molecules and

chemokine receptors involved in T cell trafficking to theintestines

IBD is associated with a massive influx of immune cellsinto the gut Previous studies indicated that increased local

10 Mediators of Inflammation

ICAM-1 P-selectin E-selectin

Relat

ive m

RNA

ratio

0

50

100

150

200

1000

1250

1500

CG

DCDG

daggerdagger

dagger

lowast

lowastlowast

lowast

lowast

lowast

Figure 8 Gene expression of endothelial adhesion molecules incolon tissues mRNA levels were analyzed by a real-time PCR TheC group was used as a calibrator and the data were presented as thefold change in gene expression relative to the calibrator Values areshown as themean plusmn SEM lowastSignificantly different from the C group(119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

secretion of proinflammatory cytokines by the inflamedcolon leads to upregulated expressions of vascular adhesionmolecules resulting in a sustained influx of inflammatorycells [26] Although inflammation predominantly occurs inthe GI tract IBD patients are likely to develop extraintestinalmanifestations [27] that may be attributed to aberrant acti-vation and homing of T cells [28] A recent study indicatedthat circulating CD4+ and CD8+ T cells are activated in bothCD and UC patients and these cells were correlated withleakage of microbial products from the impaired intestinalbarrier [5] Also the severity of DSS-induced colitis wascorrelatedwith the immune response ofMLNs [29] A similarphenomenon was also observed in this study Our resultsindicated that expressions of adhesion molecules and CCR9on Th cells and Tc cells significantly increased in both bloodand MLNs after DSS exposure These findings suggest thatboth local and systemic T cells are activated The role of Thcells in the pathogenesis of IBD has been widely studiedDysregulation ofTh cells can lead to immune cell infiltrationinto the intestinal mucosa and cause persistent inflammation[3 6] The pathogenic role of Tc cells in IBD has been lessinvestigated A previous study showed that antigen-specificTc cells caused relapsing colitis in normal mice due to thecytolytic function against the intestinal epithelium [30] Leeet al [4] reported a gene expression profile of circulatingCD8+ T cells that predicted a more aggressive disease coursefor IBD patients However the modulatory mechanism of Tccells in IBD is still under investigation

Naıve T cells constantly recirculate between the bloodand secondary lymphoid organs Once activated in secondarylymphoid organs they become effector T cells that expressadhesion molecules and chemokine receptors which controltheir extravasation into nonlymphoid tissue sites [31] T cell

trafficking to the gut and gut-associated lymphoid tissues(GALTs) requires 12057241205737 integrins The ligand MAdCAM-1is constitutively expressed by the mucosal endothelium inthe small intestine and colon [32] CCR9 is thought toparticipate in the specific localization of T cells to the smallintestines because the sole ligand for CCR9 C-C chemokineligand 25 (CCL25) is strongly expressed by the small intesti-nal epithelium [33] However Wurbel et al [34] revealedthat CCL25 expression increased during the recovery phaseafter acute DSS administration suggesting a regulatory roleof CCR9CCL25 interactions during colonic inflammationIn this study percentages of PSGL-1- LFA-1- and CCR9-expressing T cells were upregulated in acute DSS colitiswhereas expression levels of 12057241205737 integrins in colitic micedid not differ from those of normal mice It is possible thatT cell trafficking into the gut during acute DSS exposure isless dependent on 12057241205737 integrins In support of our findingsWang et al [35] reported that localization of T cells to theintestines was relatively unaffected by 12057241205737 blockade duringacute DSS-induced colitis

Gln is a critical fuel source for enterocytes and immunecells Gln supplementation attenuates gut injury by a com-plex mechanism which involves protecting the epithelialbarrier function reducing oxidative stress and modulat-ing inflammatory responses [36] The local and systemicimmunomodulatory effects of Gln have been discussed invarious experimental colitis models via different adminis-tration routes Studies using rodents with trinitrobenzenesulfonic acid-induced colitis indicated that Gln given bythe rectal route inhibits nuclear factor (NF)-120581B- and STAT-mediated inflammation in colon tissues [15] and further pre-vents colon fibrosis through downregulating gene pathwaysthat contribute to the accumulation of matrix proteins [37]We recently demonstrated that intraperitoneal pretreatmentwith alanyl-Gln a Gln-containing dipeptide widely used inparenteral nutrition suppresses cytokine expression in bloodTh cells reduces NF-120581B-mediated inflammatory responsesin the colon and upregulates expressions of genes whichpromote recovery of the colonic mucosa [17 38]

Because most exogenous Gln is absorbed in the proximalsmall intestine it might not reach the inflamed colon at asufficient concentration tomodulate inflammatory responses[36] However the enteral route of Gln administrationstill showed protective effects against DSS-induced damageOral Gln supplementation reduced the feces water contentenhanced expression of HSPs in the colonic mucosa andameliorated colon injury caused byDSS exposure [16 39 40]A previous study indicated that oral Gln attenuated leukocyteadhesion and emigration in a rodentmodel of indomethacin-induced ileitis [41] In this study we demonstrated thatoral Gln administration suppressed adhesion molecules andCCR9 expressed by T cells and downregulated the mRNAlevels of adhesion molecules expressed by endothelium incolon tissues The histological findings also support theresults that Gln administration suppressed the infiltrationof Th cells into the colon mucosa Gln consumption is animportant component of T cell activation [42 43] Differentsusceptibilities ofThandTc cells toGln supplementationmaybe explained by a higher cell population of Th cells which

Mediators of Inflammation 11

50120583m

50120583m

50120583m

50120583m

CD3

C

G

DC

DG

(a)

50120583m

50120583m

50120583m

50120583m

CD4

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 9 Representative examples of the immunofluorescence staining of T helper cells In the left column cell surface antigens were stainedfor CD3 (green FITC) and staining of CD4 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI(blue) and the last column presents a colocalized fluorescence image of CD3+CD4+ T cells

competes as a Gln source with Tc cells Further studies areneeded to investigate the molecular mechanisms involved ingene expressions of adhesion molecules and CCR9 regulatedby Gln

In conclusion this study showed for the first time thatpretreatment with oral Gln reduced adhesion molecule- andCCR9-expressing T cells induced by DSS exposure Theinhibitory abilities against adhesion molecule and CCR9expressions were more obvious inTh cells than Tc cells AlsoGln supplementation reduced gene expressions of endothe-lial adhesion molecules in colons prevented BW loss and

attenuated colon edema in coliticmice Our results imply thatdietary Gln prevented Th cell trafficking into colon tissuesand provide a new mechanism of Gln supplementation thathas beneficial effects on ameliorating the severity of acuteDSS-induced colitis

Conflict of Interests

The authors declare that they have no conflict of interests

12 Mediators of Inflammation

50120583m

CD3

50120583m

50120583m

50120583m

C

G

DC

DG

(a)

CD8

50120583m

50120583m

50120583m

50120583m

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 10 Representative immunofluorescence images of cytotoxic T cells In the left column cell surface antigens were stained for CD3(green FITC) and the staining of CD8 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI (blue)and the last column presents a colocalized fluorescence image of CD3+CD8+ T cells

Acknowledgments

The authors would like to thank Man-Hui Pai Assistant Pro-fessor of Department of Anatomy Taipei Medical Universityfor her technical assistance in immunofluorescence stainingThis studywas supported by aResearchGrant (NSC100-2320-B-038-007-MY3) from the National Science Council TaipeiTaiwan

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] C T Murphy K Nally F Shanahan and S Melgar ldquoShining alight on intestinal trafficrdquoClinical and Developmental Immunol-ogy vol 2012 Article ID 808157 14 pages 2012

Mediators of Inflammation 13

[3] L A Zenewicz A Antov and R A Flavell ldquoCD4 T-cell differ-entiation and inflammatory bowel diseaserdquo Trends inMolecularMedicine vol 15 no 5 pp 199ndash207 2009

[4] J C Lee P A Lyons E F McKinney et al ldquoGene expressionprofiling of CD8+ T cells predicts prognosis in patients withCrohn disease and ulcerative colitisrdquo Journal of Clinical Inves-tigation vol 121 no 10 pp 4170ndash4179 2011

[5] N T Funderburg S R Stubblefield Park H C Sung et alldquoCirculating CD4+ and CD8+ T cells are activated in IBDand are associated with plasma markers of inflammationrdquoImmunology vol 140 no 1 pp 87ndash97 2013

[6] E J Villablanca B Cassani U H von Andrian and J RMora ldquoBlocking lymphocyte localization to the gastrointestinalmucosa as a therapeutic strategy for inflammatory boweldiseasesrdquo Gastroenterology vol 140 no 6 pp 1776ndash1784 2011

[7] R P McEver and R D Cummings ldquoPerspectives series celladhesion in vascular biology Role of PSGL-1 binding toselectins in leukocyte recruitmentrdquo The Journal of ClinicalInvestigation vol 100 no 3 pp 485ndash491 1997

[8] C Berlin E L Berg M J Briskin et al ldquo12057241205737 Integrin medi-ates lymphocyte binding to the mucosal vascular addressinMAdCAM-1rdquo Cell vol 74 no 1 pp 185ndash195 1993

[9] K Ley C Laudanna M I Cybulsky and S Nourshargh ldquoGet-ting to the site of inflammation the leukocyte adhesion cascadeupdatedrdquoNature Reviews Immunology vol 7 no 9 pp 678ndash6892007

[10] B Moser M Wolf A Walz and P Loetscher ldquoChemokinesmultiple levels of leukocyte migration controlrdquo Trends inImmunology vol 25 no 2 pp 75ndash84 2004

[11] S Arimilli W Ferlin N Solvason S Deshpande M Howardand S Mocci ldquoChemokines in autoimmune diseasesrdquo Immuno-logical Reviews vol 177 pp 43ndash51 2000

[12] C Koenecke and R Forster ldquoCCR9 and inflammatory boweldiseaserdquo Expert Opinion on Therapeutic Targets vol 13 no 3pp 297ndash306 2009

[13] B Johansson-Lindbom and W W Agace ldquoGeneration of gut-homing T cells and their localization to the small intestinalmucosardquo Immunological Reviews vol 215 no 1 pp 226ndash2422007

[14] P EWischmeyer ldquoGlutaminemode of action in critical illnessrdquoCritical Care Medicine vol 35 no 9 pp S541ndashS544 2007

[15] N A Kretzmann H Fillmann J L Mauriz C A MarroniJ Gonzalez-Gallego and M J Tunon ldquoEffects of glutamineon proinflammatory gene expression and activation of nuclearfactor Kappa B and signal transducers and activators oftranscription in TNBS-induced colitisrdquo Inflammatory BowelDiseases vol 14 no 11 pp 1504ndash1513 2008

[16] H Xue A J D Sufit and P EWischmeyer ldquoGlutamine therapyimproves outcome of in vitro and in vivo experimental colitismodelsrdquo Journal of Parenteral and Enteral Nutrition vol 35 no2 pp 188ndash197 2011

[17] C-C Chu Y-C Hou M-H Pai C-J Chao and S-L YehldquoPretreatment with alanyl-glutamine suppresses T-helper-cell-associated cytokine expression and reduces inflammatoryresponses in mice with acute DSS-induced colitisrdquo The Journalof Nutritional Biochemistry vol 23 no 9 pp 1092ndash1099 2011

[18] S Kew S M Wells P Yaqoob F A Wallace E A Miles and PC Calder ldquoDietary glutamine enhances murine T-lymphocyteresponsivenessrdquo The Journal of Nutrition vol 129 no 8 pp1524ndash1531 1999

[19] S M Wells S Kew P Yaqoob F A Wallace and P C CalderldquoDietary glutamine enhances cytokine production by murinemacrophagesrdquo Nutrition vol 15 no 11-12 pp 881ndash884 1999

[20] Y-C Hou M-H Pai W-C Chiu Y-M Hu and S-L YehldquoEffects of dietary glutamine supplementation on lung injuryinduced by lipopolysaccharide administrationrdquo American Jour-nal of PhysiologymdashLung Cellular and Molecular Physiology vol296 no 3 pp L288ndashL295 2009

[21] M Kawada A Arihiro and E Mizoguchi ldquoInsights fromadvances in research of chemically induced experimental mod-els of human inflammatory bowel diseaserdquo World Journal ofGastroenterology vol 13 no 42 pp 5581ndash5593 2007

[22] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[23] R Yazbeck G S Howarth R N Butler M S Geier and CA Abbott ldquoBiochemical and histological changes in the smallintestine of mice with dextran sulfate sodium colitisrdquo Journal ofCellular Physiology vol 226 no 12 pp 3219ndash3224 2011

[24] M Mahler I J Bristol E H Leiter et al ldquoDifferentialsusceptibility of inbred mouse strains to dextran sulfatesodium-induced colitisrdquo American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 274 no 3 part 1 ppG544ndashG551 1998

[25] S Melgar A Karlsson and E Michaelsson ldquoAcute colitisinduced by dextran sulfate sodium progresses to chronicityin C57BL6 but not in BALBc mice correlation betweensymptoms and inflammationrdquoAmerican Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 288 no 6 pp G1328ndashG1338 2005

[26] MKoizumiN King R Lobb C Benjamin andDK PodolskyldquoExpression of vascular adhesion molecules in inflammatorybowel diseaserdquo Gastroenterology vol 103 no 3 pp 840ndash8471992

[27] L Peyrin-Biroulet E V Loftus Jr J-F Colombel and W JSandborn ldquoLong-term complications extraintestinal manifes-tations and mortality in adult Crohnrsquos disease in population-based cohortsrdquo Inflammatory Bowel Diseases vol 17 no 1 pp471ndash478 2011

[28] D H Adams and B Eksteen ldquoAberrant homing of mucosal Tcells and extra-intestinal manifestations of inflammatory boweldiseaserdquoNature Reviews Immunology vol 6 no 3 pp 244ndash2512006

[29] TW Spahn H Herbst P D Rennert et al ldquoInduction of colitisinmice deficient of Peyerrsquos patches andmesenteric lymph nodesis associated with increased disease severity and formation ofcolonic lymphoid patchesrdquo The American Journal of Pathologyvol 161 no 6 pp 2273ndash2282 2002

[30] S Nancey S Holvoet I Graber et al ldquoCD8+ Cytotoxic T CellsInduce Relapsing Colitis in Normal Micerdquo Gastroenterologyvol 131 no 2 pp 485ndash496 2006

[31] E C Butcher and L J Picker ldquoLymphocyte homing and home-ostasisrdquo Science vol 272 no 5258 pp 60ndash66 1996

[32] C Berlin R F Bargatze J J Campbell et al ldquo1205724 Integrinsmediate lymphocyte attachment and rolling under physiologicflowrdquo Cell vol 80 no 3 pp 413ndash422 1995

[33] K A Papadakis J Prehn V Nelson et al ldquoThe role of thymus-expressed chemokine and its receptor CCR9 on lymphocytesin the regional specialization of the mucosal immune systemrdquoJournal of Immunology vol 165 no 9 pp 5069ndash5076 2000

14 Mediators of Inflammation

[34] M-A Wurbel M G McIntire P Dwyer and E FiebigerldquoCCL25CCR9 interactions regulate large intestinal inflamma-tion in a murine model of acute colitisrdquo PLoS ONE vol 6 no 1Article ID e16442 2011

[35] CWang E K Hanly LWWheeler M Kaur K GMcDonaldand R D Newberry ldquoEffect of 12057241205737 blockade on intestinallymphocyte subsets and lymphoid tissue developmentrdquo Inflam-matory Bowel Diseases vol 16 no 10 pp 1751ndash1762 2010

[36] M Coeffier R Marion-Letellier and P Dechelotte ldquoPotentialfor amino acids supplementation during inflammatory boweldiseasesrdquo Inflammatory Bowel Diseases vol 16 no 3 pp 518ndash524 2010

[37] B San-Miguel I Crespo N A Kretzmann et al ldquoGlutamineprevents fibrosis development in rats with colitis induced by246-trinitrobenzene sulfonic acidrdquo The Journal of Nutritionvol 140 no 6 pp 1065ndash1071 2010

[38] Y C Hou C C Chu T L Ko et al ldquoEffects of alanyl-glutaminedipeptide on the expression of colon-inflammatory mediatorsduring the recovery phase of colitis induced by dextran sulfatesodiumrdquo European Journal of Nutrition vol 52 no 3 pp 1089ndash1098 2013

[39] Y Shiomi S Nishiumi M Ooi et al ldquoGCMS-based meta-bolomic study in mice with colitis induced by dextran sulfatesodiumrdquo Inflammatory Bowel Diseases vol 17 no 11 pp 2261ndash2274 2011

[40] M Vicario C Amat M Rivero M Moreto and C PelegrıldquoDietary glutamine affects mucosal functions in rats with mildDSS-induced colitisrdquoThe Journal of Nutrition vol 137 no 8 pp1931ndash1937 2007

[41] H Arndt F Kullmann F Reuszlig J Scholmerich and K-D Pal-itzsch ldquoGlutamine attenuates leukocyte-endothelial cell adhe-sion in indomethacin-induced intestinal inflammation in theratrdquo Journal of Parenteral and Enteral Nutrition vol 23 no 1pp 12ndash18 1999

[42] P Yaqoob and P C Calder ldquoGlutamine requirement of prolif-erating T lymphocytesrdquo Nutrition vol 13 no 7-8 pp 646ndash6511997

[43] E L Carr A Kelman G S Wu et al ldquoGlutamine uptake andmetabolism are coordinately regulated by ERKMAPK duringT lymphocyte activationrdquo Journal of Immunology vol 185 no2 pp 1037ndash1044 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 9

C G DC DG

PSG

L-1

()

0

10

20

30

40

50

lowastlowast

(a)

CD11

a (

)

C G DC DG0

5

10

15

20

25

CD11

a (

)

12057241205737

inte

grin

s (

)

(b)

C G DC DG0

5

10

15

20

25

CD11

a (

)

dagger

lowast

(c)

CCR9

()

C G DC DG0

5

10

15

20

25

lowast

lowast

(d)

CCR

9 ex

pres

sing

0

15

30

45

60

lowastlowast

CD+ 812057241205737+

T ce

lls (

)

C G DC DG

(e)

Figure 7 Percentage of adhesion molecule- and chemokine receptor-expressing cytotoxic T (Tc) cells in mesenteric lymph nodes (MLNs)CD8-positiveMLN lymphocytes were gated to analyze expressions of PSGL-1 12057241205737 integrins CD11a and CCR9 by flow cytometry ((a)ndash(d))(e) Expression of CCR9 by CD8 and 12057241205737 integrin double-positive MLN lymphocytes Values are shown as the mean plusmn SEM lowastSignificantlydifferent from the C group (119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

migration to the colon plays an important role in theprogression to chronicity [25] Regarding the high sensitivityto DSS C57BL6 mice were used in this study to analyze theconsequences of DSS exposure on adhesion molecules and

chemokine receptors involved in T cell trafficking to theintestines

IBD is associated with a massive influx of immune cellsinto the gut Previous studies indicated that increased local

10 Mediators of Inflammation

ICAM-1 P-selectin E-selectin

Relat

ive m

RNA

ratio

0

50

100

150

200

1000

1250

1500

CG

DCDG

daggerdagger

dagger

lowast

lowastlowast

lowast

lowast

lowast

Figure 8 Gene expression of endothelial adhesion molecules incolon tissues mRNA levels were analyzed by a real-time PCR TheC group was used as a calibrator and the data were presented as thefold change in gene expression relative to the calibrator Values areshown as themean plusmn SEM lowastSignificantly different from the C group(119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

secretion of proinflammatory cytokines by the inflamedcolon leads to upregulated expressions of vascular adhesionmolecules resulting in a sustained influx of inflammatorycells [26] Although inflammation predominantly occurs inthe GI tract IBD patients are likely to develop extraintestinalmanifestations [27] that may be attributed to aberrant acti-vation and homing of T cells [28] A recent study indicatedthat circulating CD4+ and CD8+ T cells are activated in bothCD and UC patients and these cells were correlated withleakage of microbial products from the impaired intestinalbarrier [5] Also the severity of DSS-induced colitis wascorrelatedwith the immune response ofMLNs [29] A similarphenomenon was also observed in this study Our resultsindicated that expressions of adhesion molecules and CCR9on Th cells and Tc cells significantly increased in both bloodand MLNs after DSS exposure These findings suggest thatboth local and systemic T cells are activated The role of Thcells in the pathogenesis of IBD has been widely studiedDysregulation ofTh cells can lead to immune cell infiltrationinto the intestinal mucosa and cause persistent inflammation[3 6] The pathogenic role of Tc cells in IBD has been lessinvestigated A previous study showed that antigen-specificTc cells caused relapsing colitis in normal mice due to thecytolytic function against the intestinal epithelium [30] Leeet al [4] reported a gene expression profile of circulatingCD8+ T cells that predicted a more aggressive disease coursefor IBD patients However the modulatory mechanism of Tccells in IBD is still under investigation

Naıve T cells constantly recirculate between the bloodand secondary lymphoid organs Once activated in secondarylymphoid organs they become effector T cells that expressadhesion molecules and chemokine receptors which controltheir extravasation into nonlymphoid tissue sites [31] T cell

trafficking to the gut and gut-associated lymphoid tissues(GALTs) requires 12057241205737 integrins The ligand MAdCAM-1is constitutively expressed by the mucosal endothelium inthe small intestine and colon [32] CCR9 is thought toparticipate in the specific localization of T cells to the smallintestines because the sole ligand for CCR9 C-C chemokineligand 25 (CCL25) is strongly expressed by the small intesti-nal epithelium [33] However Wurbel et al [34] revealedthat CCL25 expression increased during the recovery phaseafter acute DSS administration suggesting a regulatory roleof CCR9CCL25 interactions during colonic inflammationIn this study percentages of PSGL-1- LFA-1- and CCR9-expressing T cells were upregulated in acute DSS colitiswhereas expression levels of 12057241205737 integrins in colitic micedid not differ from those of normal mice It is possible thatT cell trafficking into the gut during acute DSS exposure isless dependent on 12057241205737 integrins In support of our findingsWang et al [35] reported that localization of T cells to theintestines was relatively unaffected by 12057241205737 blockade duringacute DSS-induced colitis

Gln is a critical fuel source for enterocytes and immunecells Gln supplementation attenuates gut injury by a com-plex mechanism which involves protecting the epithelialbarrier function reducing oxidative stress and modulat-ing inflammatory responses [36] The local and systemicimmunomodulatory effects of Gln have been discussed invarious experimental colitis models via different adminis-tration routes Studies using rodents with trinitrobenzenesulfonic acid-induced colitis indicated that Gln given bythe rectal route inhibits nuclear factor (NF)-120581B- and STAT-mediated inflammation in colon tissues [15] and further pre-vents colon fibrosis through downregulating gene pathwaysthat contribute to the accumulation of matrix proteins [37]We recently demonstrated that intraperitoneal pretreatmentwith alanyl-Gln a Gln-containing dipeptide widely used inparenteral nutrition suppresses cytokine expression in bloodTh cells reduces NF-120581B-mediated inflammatory responsesin the colon and upregulates expressions of genes whichpromote recovery of the colonic mucosa [17 38]

Because most exogenous Gln is absorbed in the proximalsmall intestine it might not reach the inflamed colon at asufficient concentration tomodulate inflammatory responses[36] However the enteral route of Gln administrationstill showed protective effects against DSS-induced damageOral Gln supplementation reduced the feces water contentenhanced expression of HSPs in the colonic mucosa andameliorated colon injury caused byDSS exposure [16 39 40]A previous study indicated that oral Gln attenuated leukocyteadhesion and emigration in a rodentmodel of indomethacin-induced ileitis [41] In this study we demonstrated thatoral Gln administration suppressed adhesion molecules andCCR9 expressed by T cells and downregulated the mRNAlevels of adhesion molecules expressed by endothelium incolon tissues The histological findings also support theresults that Gln administration suppressed the infiltrationof Th cells into the colon mucosa Gln consumption is animportant component of T cell activation [42 43] Differentsusceptibilities ofThandTc cells toGln supplementationmaybe explained by a higher cell population of Th cells which

Mediators of Inflammation 11

50120583m

50120583m

50120583m

50120583m

CD3

C

G

DC

DG

(a)

50120583m

50120583m

50120583m

50120583m

CD4

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 9 Representative examples of the immunofluorescence staining of T helper cells In the left column cell surface antigens were stainedfor CD3 (green FITC) and staining of CD4 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI(blue) and the last column presents a colocalized fluorescence image of CD3+CD4+ T cells

competes as a Gln source with Tc cells Further studies areneeded to investigate the molecular mechanisms involved ingene expressions of adhesion molecules and CCR9 regulatedby Gln

In conclusion this study showed for the first time thatpretreatment with oral Gln reduced adhesion molecule- andCCR9-expressing T cells induced by DSS exposure Theinhibitory abilities against adhesion molecule and CCR9expressions were more obvious inTh cells than Tc cells AlsoGln supplementation reduced gene expressions of endothe-lial adhesion molecules in colons prevented BW loss and

attenuated colon edema in coliticmice Our results imply thatdietary Gln prevented Th cell trafficking into colon tissuesand provide a new mechanism of Gln supplementation thathas beneficial effects on ameliorating the severity of acuteDSS-induced colitis

Conflict of Interests

The authors declare that they have no conflict of interests

12 Mediators of Inflammation

50120583m

CD3

50120583m

50120583m

50120583m

C

G

DC

DG

(a)

CD8

50120583m

50120583m

50120583m

50120583m

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 10 Representative immunofluorescence images of cytotoxic T cells In the left column cell surface antigens were stained for CD3(green FITC) and the staining of CD8 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI (blue)and the last column presents a colocalized fluorescence image of CD3+CD8+ T cells

Acknowledgments

The authors would like to thank Man-Hui Pai Assistant Pro-fessor of Department of Anatomy Taipei Medical Universityfor her technical assistance in immunofluorescence stainingThis studywas supported by aResearchGrant (NSC100-2320-B-038-007-MY3) from the National Science Council TaipeiTaiwan

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] C T Murphy K Nally F Shanahan and S Melgar ldquoShining alight on intestinal trafficrdquoClinical and Developmental Immunol-ogy vol 2012 Article ID 808157 14 pages 2012

Mediators of Inflammation 13

[3] L A Zenewicz A Antov and R A Flavell ldquoCD4 T-cell differ-entiation and inflammatory bowel diseaserdquo Trends inMolecularMedicine vol 15 no 5 pp 199ndash207 2009

[4] J C Lee P A Lyons E F McKinney et al ldquoGene expressionprofiling of CD8+ T cells predicts prognosis in patients withCrohn disease and ulcerative colitisrdquo Journal of Clinical Inves-tigation vol 121 no 10 pp 4170ndash4179 2011

[5] N T Funderburg S R Stubblefield Park H C Sung et alldquoCirculating CD4+ and CD8+ T cells are activated in IBDand are associated with plasma markers of inflammationrdquoImmunology vol 140 no 1 pp 87ndash97 2013

[6] E J Villablanca B Cassani U H von Andrian and J RMora ldquoBlocking lymphocyte localization to the gastrointestinalmucosa as a therapeutic strategy for inflammatory boweldiseasesrdquo Gastroenterology vol 140 no 6 pp 1776ndash1784 2011

[7] R P McEver and R D Cummings ldquoPerspectives series celladhesion in vascular biology Role of PSGL-1 binding toselectins in leukocyte recruitmentrdquo The Journal of ClinicalInvestigation vol 100 no 3 pp 485ndash491 1997

[8] C Berlin E L Berg M J Briskin et al ldquo12057241205737 Integrin medi-ates lymphocyte binding to the mucosal vascular addressinMAdCAM-1rdquo Cell vol 74 no 1 pp 185ndash195 1993

[9] K Ley C Laudanna M I Cybulsky and S Nourshargh ldquoGet-ting to the site of inflammation the leukocyte adhesion cascadeupdatedrdquoNature Reviews Immunology vol 7 no 9 pp 678ndash6892007

[10] B Moser M Wolf A Walz and P Loetscher ldquoChemokinesmultiple levels of leukocyte migration controlrdquo Trends inImmunology vol 25 no 2 pp 75ndash84 2004

[11] S Arimilli W Ferlin N Solvason S Deshpande M Howardand S Mocci ldquoChemokines in autoimmune diseasesrdquo Immuno-logical Reviews vol 177 pp 43ndash51 2000

[12] C Koenecke and R Forster ldquoCCR9 and inflammatory boweldiseaserdquo Expert Opinion on Therapeutic Targets vol 13 no 3pp 297ndash306 2009

[13] B Johansson-Lindbom and W W Agace ldquoGeneration of gut-homing T cells and their localization to the small intestinalmucosardquo Immunological Reviews vol 215 no 1 pp 226ndash2422007

[14] P EWischmeyer ldquoGlutaminemode of action in critical illnessrdquoCritical Care Medicine vol 35 no 9 pp S541ndashS544 2007

[15] N A Kretzmann H Fillmann J L Mauriz C A MarroniJ Gonzalez-Gallego and M J Tunon ldquoEffects of glutamineon proinflammatory gene expression and activation of nuclearfactor Kappa B and signal transducers and activators oftranscription in TNBS-induced colitisrdquo Inflammatory BowelDiseases vol 14 no 11 pp 1504ndash1513 2008

[16] H Xue A J D Sufit and P EWischmeyer ldquoGlutamine therapyimproves outcome of in vitro and in vivo experimental colitismodelsrdquo Journal of Parenteral and Enteral Nutrition vol 35 no2 pp 188ndash197 2011

[17] C-C Chu Y-C Hou M-H Pai C-J Chao and S-L YehldquoPretreatment with alanyl-glutamine suppresses T-helper-cell-associated cytokine expression and reduces inflammatoryresponses in mice with acute DSS-induced colitisrdquo The Journalof Nutritional Biochemistry vol 23 no 9 pp 1092ndash1099 2011

[18] S Kew S M Wells P Yaqoob F A Wallace E A Miles and PC Calder ldquoDietary glutamine enhances murine T-lymphocyteresponsivenessrdquo The Journal of Nutrition vol 129 no 8 pp1524ndash1531 1999

[19] S M Wells S Kew P Yaqoob F A Wallace and P C CalderldquoDietary glutamine enhances cytokine production by murinemacrophagesrdquo Nutrition vol 15 no 11-12 pp 881ndash884 1999

[20] Y-C Hou M-H Pai W-C Chiu Y-M Hu and S-L YehldquoEffects of dietary glutamine supplementation on lung injuryinduced by lipopolysaccharide administrationrdquo American Jour-nal of PhysiologymdashLung Cellular and Molecular Physiology vol296 no 3 pp L288ndashL295 2009

[21] M Kawada A Arihiro and E Mizoguchi ldquoInsights fromadvances in research of chemically induced experimental mod-els of human inflammatory bowel diseaserdquo World Journal ofGastroenterology vol 13 no 42 pp 5581ndash5593 2007

[22] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[23] R Yazbeck G S Howarth R N Butler M S Geier and CA Abbott ldquoBiochemical and histological changes in the smallintestine of mice with dextran sulfate sodium colitisrdquo Journal ofCellular Physiology vol 226 no 12 pp 3219ndash3224 2011

[24] M Mahler I J Bristol E H Leiter et al ldquoDifferentialsusceptibility of inbred mouse strains to dextran sulfatesodium-induced colitisrdquo American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 274 no 3 part 1 ppG544ndashG551 1998

[25] S Melgar A Karlsson and E Michaelsson ldquoAcute colitisinduced by dextran sulfate sodium progresses to chronicityin C57BL6 but not in BALBc mice correlation betweensymptoms and inflammationrdquoAmerican Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 288 no 6 pp G1328ndashG1338 2005

[26] MKoizumiN King R Lobb C Benjamin andDK PodolskyldquoExpression of vascular adhesion molecules in inflammatorybowel diseaserdquo Gastroenterology vol 103 no 3 pp 840ndash8471992

[27] L Peyrin-Biroulet E V Loftus Jr J-F Colombel and W JSandborn ldquoLong-term complications extraintestinal manifes-tations and mortality in adult Crohnrsquos disease in population-based cohortsrdquo Inflammatory Bowel Diseases vol 17 no 1 pp471ndash478 2011

[28] D H Adams and B Eksteen ldquoAberrant homing of mucosal Tcells and extra-intestinal manifestations of inflammatory boweldiseaserdquoNature Reviews Immunology vol 6 no 3 pp 244ndash2512006

[29] TW Spahn H Herbst P D Rennert et al ldquoInduction of colitisinmice deficient of Peyerrsquos patches andmesenteric lymph nodesis associated with increased disease severity and formation ofcolonic lymphoid patchesrdquo The American Journal of Pathologyvol 161 no 6 pp 2273ndash2282 2002

[30] S Nancey S Holvoet I Graber et al ldquoCD8+ Cytotoxic T CellsInduce Relapsing Colitis in Normal Micerdquo Gastroenterologyvol 131 no 2 pp 485ndash496 2006

[31] E C Butcher and L J Picker ldquoLymphocyte homing and home-ostasisrdquo Science vol 272 no 5258 pp 60ndash66 1996

[32] C Berlin R F Bargatze J J Campbell et al ldquo1205724 Integrinsmediate lymphocyte attachment and rolling under physiologicflowrdquo Cell vol 80 no 3 pp 413ndash422 1995

[33] K A Papadakis J Prehn V Nelson et al ldquoThe role of thymus-expressed chemokine and its receptor CCR9 on lymphocytesin the regional specialization of the mucosal immune systemrdquoJournal of Immunology vol 165 no 9 pp 5069ndash5076 2000

14 Mediators of Inflammation

[34] M-A Wurbel M G McIntire P Dwyer and E FiebigerldquoCCL25CCR9 interactions regulate large intestinal inflamma-tion in a murine model of acute colitisrdquo PLoS ONE vol 6 no 1Article ID e16442 2011

[35] CWang E K Hanly LWWheeler M Kaur K GMcDonaldand R D Newberry ldquoEffect of 12057241205737 blockade on intestinallymphocyte subsets and lymphoid tissue developmentrdquo Inflam-matory Bowel Diseases vol 16 no 10 pp 1751ndash1762 2010

[36] M Coeffier R Marion-Letellier and P Dechelotte ldquoPotentialfor amino acids supplementation during inflammatory boweldiseasesrdquo Inflammatory Bowel Diseases vol 16 no 3 pp 518ndash524 2010

[37] B San-Miguel I Crespo N A Kretzmann et al ldquoGlutamineprevents fibrosis development in rats with colitis induced by246-trinitrobenzene sulfonic acidrdquo The Journal of Nutritionvol 140 no 6 pp 1065ndash1071 2010

[38] Y C Hou C C Chu T L Ko et al ldquoEffects of alanyl-glutaminedipeptide on the expression of colon-inflammatory mediatorsduring the recovery phase of colitis induced by dextran sulfatesodiumrdquo European Journal of Nutrition vol 52 no 3 pp 1089ndash1098 2013

[39] Y Shiomi S Nishiumi M Ooi et al ldquoGCMS-based meta-bolomic study in mice with colitis induced by dextran sulfatesodiumrdquo Inflammatory Bowel Diseases vol 17 no 11 pp 2261ndash2274 2011

[40] M Vicario C Amat M Rivero M Moreto and C PelegrıldquoDietary glutamine affects mucosal functions in rats with mildDSS-induced colitisrdquoThe Journal of Nutrition vol 137 no 8 pp1931ndash1937 2007

[41] H Arndt F Kullmann F Reuszlig J Scholmerich and K-D Pal-itzsch ldquoGlutamine attenuates leukocyte-endothelial cell adhe-sion in indomethacin-induced intestinal inflammation in theratrdquo Journal of Parenteral and Enteral Nutrition vol 23 no 1pp 12ndash18 1999

[42] P Yaqoob and P C Calder ldquoGlutamine requirement of prolif-erating T lymphocytesrdquo Nutrition vol 13 no 7-8 pp 646ndash6511997

[43] E L Carr A Kelman G S Wu et al ldquoGlutamine uptake andmetabolism are coordinately regulated by ERKMAPK duringT lymphocyte activationrdquo Journal of Immunology vol 185 no2 pp 1037ndash1044 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Mediators of Inflammation

ICAM-1 P-selectin E-selectin

Relat

ive m

RNA

ratio

0

50

100

150

200

1000

1250

1500

CG

DCDG

daggerdagger

dagger

lowast

lowastlowast

lowast

lowast

lowast

Figure 8 Gene expression of endothelial adhesion molecules incolon tissues mRNA levels were analyzed by a real-time PCR TheC group was used as a calibrator and the data were presented as thefold change in gene expression relative to the calibrator Values areshown as themean plusmn SEM lowastSignificantly different from the C group(119875 lt 005) daggerSignificantly different from the DC group (119875 lt 005)

secretion of proinflammatory cytokines by the inflamedcolon leads to upregulated expressions of vascular adhesionmolecules resulting in a sustained influx of inflammatorycells [26] Although inflammation predominantly occurs inthe GI tract IBD patients are likely to develop extraintestinalmanifestations [27] that may be attributed to aberrant acti-vation and homing of T cells [28] A recent study indicatedthat circulating CD4+ and CD8+ T cells are activated in bothCD and UC patients and these cells were correlated withleakage of microbial products from the impaired intestinalbarrier [5] Also the severity of DSS-induced colitis wascorrelatedwith the immune response ofMLNs [29] A similarphenomenon was also observed in this study Our resultsindicated that expressions of adhesion molecules and CCR9on Th cells and Tc cells significantly increased in both bloodand MLNs after DSS exposure These findings suggest thatboth local and systemic T cells are activated The role of Thcells in the pathogenesis of IBD has been widely studiedDysregulation ofTh cells can lead to immune cell infiltrationinto the intestinal mucosa and cause persistent inflammation[3 6] The pathogenic role of Tc cells in IBD has been lessinvestigated A previous study showed that antigen-specificTc cells caused relapsing colitis in normal mice due to thecytolytic function against the intestinal epithelium [30] Leeet al [4] reported a gene expression profile of circulatingCD8+ T cells that predicted a more aggressive disease coursefor IBD patients However the modulatory mechanism of Tccells in IBD is still under investigation

Naıve T cells constantly recirculate between the bloodand secondary lymphoid organs Once activated in secondarylymphoid organs they become effector T cells that expressadhesion molecules and chemokine receptors which controltheir extravasation into nonlymphoid tissue sites [31] T cell

trafficking to the gut and gut-associated lymphoid tissues(GALTs) requires 12057241205737 integrins The ligand MAdCAM-1is constitutively expressed by the mucosal endothelium inthe small intestine and colon [32] CCR9 is thought toparticipate in the specific localization of T cells to the smallintestines because the sole ligand for CCR9 C-C chemokineligand 25 (CCL25) is strongly expressed by the small intesti-nal epithelium [33] However Wurbel et al [34] revealedthat CCL25 expression increased during the recovery phaseafter acute DSS administration suggesting a regulatory roleof CCR9CCL25 interactions during colonic inflammationIn this study percentages of PSGL-1- LFA-1- and CCR9-expressing T cells were upregulated in acute DSS colitiswhereas expression levels of 12057241205737 integrins in colitic micedid not differ from those of normal mice It is possible thatT cell trafficking into the gut during acute DSS exposure isless dependent on 12057241205737 integrins In support of our findingsWang et al [35] reported that localization of T cells to theintestines was relatively unaffected by 12057241205737 blockade duringacute DSS-induced colitis

Gln is a critical fuel source for enterocytes and immunecells Gln supplementation attenuates gut injury by a com-plex mechanism which involves protecting the epithelialbarrier function reducing oxidative stress and modulat-ing inflammatory responses [36] The local and systemicimmunomodulatory effects of Gln have been discussed invarious experimental colitis models via different adminis-tration routes Studies using rodents with trinitrobenzenesulfonic acid-induced colitis indicated that Gln given bythe rectal route inhibits nuclear factor (NF)-120581B- and STAT-mediated inflammation in colon tissues [15] and further pre-vents colon fibrosis through downregulating gene pathwaysthat contribute to the accumulation of matrix proteins [37]We recently demonstrated that intraperitoneal pretreatmentwith alanyl-Gln a Gln-containing dipeptide widely used inparenteral nutrition suppresses cytokine expression in bloodTh cells reduces NF-120581B-mediated inflammatory responsesin the colon and upregulates expressions of genes whichpromote recovery of the colonic mucosa [17 38]

Because most exogenous Gln is absorbed in the proximalsmall intestine it might not reach the inflamed colon at asufficient concentration tomodulate inflammatory responses[36] However the enteral route of Gln administrationstill showed protective effects against DSS-induced damageOral Gln supplementation reduced the feces water contentenhanced expression of HSPs in the colonic mucosa andameliorated colon injury caused byDSS exposure [16 39 40]A previous study indicated that oral Gln attenuated leukocyteadhesion and emigration in a rodentmodel of indomethacin-induced ileitis [41] In this study we demonstrated thatoral Gln administration suppressed adhesion molecules andCCR9 expressed by T cells and downregulated the mRNAlevels of adhesion molecules expressed by endothelium incolon tissues The histological findings also support theresults that Gln administration suppressed the infiltrationof Th cells into the colon mucosa Gln consumption is animportant component of T cell activation [42 43] Differentsusceptibilities ofThandTc cells toGln supplementationmaybe explained by a higher cell population of Th cells which

Mediators of Inflammation 11

50120583m

50120583m

50120583m

50120583m

CD3

C

G

DC

DG

(a)

50120583m

50120583m

50120583m

50120583m

CD4

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 9 Representative examples of the immunofluorescence staining of T helper cells In the left column cell surface antigens were stainedfor CD3 (green FITC) and staining of CD4 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI(blue) and the last column presents a colocalized fluorescence image of CD3+CD4+ T cells

competes as a Gln source with Tc cells Further studies areneeded to investigate the molecular mechanisms involved ingene expressions of adhesion molecules and CCR9 regulatedby Gln

In conclusion this study showed for the first time thatpretreatment with oral Gln reduced adhesion molecule- andCCR9-expressing T cells induced by DSS exposure Theinhibitory abilities against adhesion molecule and CCR9expressions were more obvious inTh cells than Tc cells AlsoGln supplementation reduced gene expressions of endothe-lial adhesion molecules in colons prevented BW loss and

attenuated colon edema in coliticmice Our results imply thatdietary Gln prevented Th cell trafficking into colon tissuesand provide a new mechanism of Gln supplementation thathas beneficial effects on ameliorating the severity of acuteDSS-induced colitis

Conflict of Interests

The authors declare that they have no conflict of interests

12 Mediators of Inflammation

50120583m

CD3

50120583m

50120583m

50120583m

C

G

DC

DG

(a)

CD8

50120583m

50120583m

50120583m

50120583m

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 10 Representative immunofluorescence images of cytotoxic T cells In the left column cell surface antigens were stained for CD3(green FITC) and the staining of CD8 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI (blue)and the last column presents a colocalized fluorescence image of CD3+CD8+ T cells

Acknowledgments

The authors would like to thank Man-Hui Pai Assistant Pro-fessor of Department of Anatomy Taipei Medical Universityfor her technical assistance in immunofluorescence stainingThis studywas supported by aResearchGrant (NSC100-2320-B-038-007-MY3) from the National Science Council TaipeiTaiwan

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] C T Murphy K Nally F Shanahan and S Melgar ldquoShining alight on intestinal trafficrdquoClinical and Developmental Immunol-ogy vol 2012 Article ID 808157 14 pages 2012

Mediators of Inflammation 13

[3] L A Zenewicz A Antov and R A Flavell ldquoCD4 T-cell differ-entiation and inflammatory bowel diseaserdquo Trends inMolecularMedicine vol 15 no 5 pp 199ndash207 2009

[4] J C Lee P A Lyons E F McKinney et al ldquoGene expressionprofiling of CD8+ T cells predicts prognosis in patients withCrohn disease and ulcerative colitisrdquo Journal of Clinical Inves-tigation vol 121 no 10 pp 4170ndash4179 2011

[5] N T Funderburg S R Stubblefield Park H C Sung et alldquoCirculating CD4+ and CD8+ T cells are activated in IBDand are associated with plasma markers of inflammationrdquoImmunology vol 140 no 1 pp 87ndash97 2013

[6] E J Villablanca B Cassani U H von Andrian and J RMora ldquoBlocking lymphocyte localization to the gastrointestinalmucosa as a therapeutic strategy for inflammatory boweldiseasesrdquo Gastroenterology vol 140 no 6 pp 1776ndash1784 2011

[7] R P McEver and R D Cummings ldquoPerspectives series celladhesion in vascular biology Role of PSGL-1 binding toselectins in leukocyte recruitmentrdquo The Journal of ClinicalInvestigation vol 100 no 3 pp 485ndash491 1997

[8] C Berlin E L Berg M J Briskin et al ldquo12057241205737 Integrin medi-ates lymphocyte binding to the mucosal vascular addressinMAdCAM-1rdquo Cell vol 74 no 1 pp 185ndash195 1993

[9] K Ley C Laudanna M I Cybulsky and S Nourshargh ldquoGet-ting to the site of inflammation the leukocyte adhesion cascadeupdatedrdquoNature Reviews Immunology vol 7 no 9 pp 678ndash6892007

[10] B Moser M Wolf A Walz and P Loetscher ldquoChemokinesmultiple levels of leukocyte migration controlrdquo Trends inImmunology vol 25 no 2 pp 75ndash84 2004

[11] S Arimilli W Ferlin N Solvason S Deshpande M Howardand S Mocci ldquoChemokines in autoimmune diseasesrdquo Immuno-logical Reviews vol 177 pp 43ndash51 2000

[12] C Koenecke and R Forster ldquoCCR9 and inflammatory boweldiseaserdquo Expert Opinion on Therapeutic Targets vol 13 no 3pp 297ndash306 2009

[13] B Johansson-Lindbom and W W Agace ldquoGeneration of gut-homing T cells and their localization to the small intestinalmucosardquo Immunological Reviews vol 215 no 1 pp 226ndash2422007

[14] P EWischmeyer ldquoGlutaminemode of action in critical illnessrdquoCritical Care Medicine vol 35 no 9 pp S541ndashS544 2007

[15] N A Kretzmann H Fillmann J L Mauriz C A MarroniJ Gonzalez-Gallego and M J Tunon ldquoEffects of glutamineon proinflammatory gene expression and activation of nuclearfactor Kappa B and signal transducers and activators oftranscription in TNBS-induced colitisrdquo Inflammatory BowelDiseases vol 14 no 11 pp 1504ndash1513 2008

[16] H Xue A J D Sufit and P EWischmeyer ldquoGlutamine therapyimproves outcome of in vitro and in vivo experimental colitismodelsrdquo Journal of Parenteral and Enteral Nutrition vol 35 no2 pp 188ndash197 2011

[17] C-C Chu Y-C Hou M-H Pai C-J Chao and S-L YehldquoPretreatment with alanyl-glutamine suppresses T-helper-cell-associated cytokine expression and reduces inflammatoryresponses in mice with acute DSS-induced colitisrdquo The Journalof Nutritional Biochemistry vol 23 no 9 pp 1092ndash1099 2011

[18] S Kew S M Wells P Yaqoob F A Wallace E A Miles and PC Calder ldquoDietary glutamine enhances murine T-lymphocyteresponsivenessrdquo The Journal of Nutrition vol 129 no 8 pp1524ndash1531 1999

[19] S M Wells S Kew P Yaqoob F A Wallace and P C CalderldquoDietary glutamine enhances cytokine production by murinemacrophagesrdquo Nutrition vol 15 no 11-12 pp 881ndash884 1999

[20] Y-C Hou M-H Pai W-C Chiu Y-M Hu and S-L YehldquoEffects of dietary glutamine supplementation on lung injuryinduced by lipopolysaccharide administrationrdquo American Jour-nal of PhysiologymdashLung Cellular and Molecular Physiology vol296 no 3 pp L288ndashL295 2009

[21] M Kawada A Arihiro and E Mizoguchi ldquoInsights fromadvances in research of chemically induced experimental mod-els of human inflammatory bowel diseaserdquo World Journal ofGastroenterology vol 13 no 42 pp 5581ndash5593 2007

[22] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[23] R Yazbeck G S Howarth R N Butler M S Geier and CA Abbott ldquoBiochemical and histological changes in the smallintestine of mice with dextran sulfate sodium colitisrdquo Journal ofCellular Physiology vol 226 no 12 pp 3219ndash3224 2011

[24] M Mahler I J Bristol E H Leiter et al ldquoDifferentialsusceptibility of inbred mouse strains to dextran sulfatesodium-induced colitisrdquo American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 274 no 3 part 1 ppG544ndashG551 1998

[25] S Melgar A Karlsson and E Michaelsson ldquoAcute colitisinduced by dextran sulfate sodium progresses to chronicityin C57BL6 but not in BALBc mice correlation betweensymptoms and inflammationrdquoAmerican Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 288 no 6 pp G1328ndashG1338 2005

[26] MKoizumiN King R Lobb C Benjamin andDK PodolskyldquoExpression of vascular adhesion molecules in inflammatorybowel diseaserdquo Gastroenterology vol 103 no 3 pp 840ndash8471992

[27] L Peyrin-Biroulet E V Loftus Jr J-F Colombel and W JSandborn ldquoLong-term complications extraintestinal manifes-tations and mortality in adult Crohnrsquos disease in population-based cohortsrdquo Inflammatory Bowel Diseases vol 17 no 1 pp471ndash478 2011

[28] D H Adams and B Eksteen ldquoAberrant homing of mucosal Tcells and extra-intestinal manifestations of inflammatory boweldiseaserdquoNature Reviews Immunology vol 6 no 3 pp 244ndash2512006

[29] TW Spahn H Herbst P D Rennert et al ldquoInduction of colitisinmice deficient of Peyerrsquos patches andmesenteric lymph nodesis associated with increased disease severity and formation ofcolonic lymphoid patchesrdquo The American Journal of Pathologyvol 161 no 6 pp 2273ndash2282 2002

[30] S Nancey S Holvoet I Graber et al ldquoCD8+ Cytotoxic T CellsInduce Relapsing Colitis in Normal Micerdquo Gastroenterologyvol 131 no 2 pp 485ndash496 2006

[31] E C Butcher and L J Picker ldquoLymphocyte homing and home-ostasisrdquo Science vol 272 no 5258 pp 60ndash66 1996

[32] C Berlin R F Bargatze J J Campbell et al ldquo1205724 Integrinsmediate lymphocyte attachment and rolling under physiologicflowrdquo Cell vol 80 no 3 pp 413ndash422 1995

[33] K A Papadakis J Prehn V Nelson et al ldquoThe role of thymus-expressed chemokine and its receptor CCR9 on lymphocytesin the regional specialization of the mucosal immune systemrdquoJournal of Immunology vol 165 no 9 pp 5069ndash5076 2000

14 Mediators of Inflammation

[34] M-A Wurbel M G McIntire P Dwyer and E FiebigerldquoCCL25CCR9 interactions regulate large intestinal inflamma-tion in a murine model of acute colitisrdquo PLoS ONE vol 6 no 1Article ID e16442 2011

[35] CWang E K Hanly LWWheeler M Kaur K GMcDonaldand R D Newberry ldquoEffect of 12057241205737 blockade on intestinallymphocyte subsets and lymphoid tissue developmentrdquo Inflam-matory Bowel Diseases vol 16 no 10 pp 1751ndash1762 2010

[36] M Coeffier R Marion-Letellier and P Dechelotte ldquoPotentialfor amino acids supplementation during inflammatory boweldiseasesrdquo Inflammatory Bowel Diseases vol 16 no 3 pp 518ndash524 2010

[37] B San-Miguel I Crespo N A Kretzmann et al ldquoGlutamineprevents fibrosis development in rats with colitis induced by246-trinitrobenzene sulfonic acidrdquo The Journal of Nutritionvol 140 no 6 pp 1065ndash1071 2010

[38] Y C Hou C C Chu T L Ko et al ldquoEffects of alanyl-glutaminedipeptide on the expression of colon-inflammatory mediatorsduring the recovery phase of colitis induced by dextran sulfatesodiumrdquo European Journal of Nutrition vol 52 no 3 pp 1089ndash1098 2013

[39] Y Shiomi S Nishiumi M Ooi et al ldquoGCMS-based meta-bolomic study in mice with colitis induced by dextran sulfatesodiumrdquo Inflammatory Bowel Diseases vol 17 no 11 pp 2261ndash2274 2011

[40] M Vicario C Amat M Rivero M Moreto and C PelegrıldquoDietary glutamine affects mucosal functions in rats with mildDSS-induced colitisrdquoThe Journal of Nutrition vol 137 no 8 pp1931ndash1937 2007

[41] H Arndt F Kullmann F Reuszlig J Scholmerich and K-D Pal-itzsch ldquoGlutamine attenuates leukocyte-endothelial cell adhe-sion in indomethacin-induced intestinal inflammation in theratrdquo Journal of Parenteral and Enteral Nutrition vol 23 no 1pp 12ndash18 1999

[42] P Yaqoob and P C Calder ldquoGlutamine requirement of prolif-erating T lymphocytesrdquo Nutrition vol 13 no 7-8 pp 646ndash6511997

[43] E L Carr A Kelman G S Wu et al ldquoGlutamine uptake andmetabolism are coordinately regulated by ERKMAPK duringT lymphocyte activationrdquo Journal of Immunology vol 185 no2 pp 1037ndash1044 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 11

50120583m

50120583m

50120583m

50120583m

CD3

C

G

DC

DG

(a)

50120583m

50120583m

50120583m

50120583m

CD4

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 9 Representative examples of the immunofluorescence staining of T helper cells In the left column cell surface antigens were stainedfor CD3 (green FITC) and staining of CD4 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI(blue) and the last column presents a colocalized fluorescence image of CD3+CD4+ T cells

competes as a Gln source with Tc cells Further studies areneeded to investigate the molecular mechanisms involved ingene expressions of adhesion molecules and CCR9 regulatedby Gln

In conclusion this study showed for the first time thatpretreatment with oral Gln reduced adhesion molecule- andCCR9-expressing T cells induced by DSS exposure Theinhibitory abilities against adhesion molecule and CCR9expressions were more obvious inTh cells than Tc cells AlsoGln supplementation reduced gene expressions of endothe-lial adhesion molecules in colons prevented BW loss and

attenuated colon edema in coliticmice Our results imply thatdietary Gln prevented Th cell trafficking into colon tissuesand provide a new mechanism of Gln supplementation thathas beneficial effects on ameliorating the severity of acuteDSS-induced colitis

Conflict of Interests

The authors declare that they have no conflict of interests

12 Mediators of Inflammation

50120583m

CD3

50120583m

50120583m

50120583m

C

G

DC

DG

(a)

CD8

50120583m

50120583m

50120583m

50120583m

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 10 Representative immunofluorescence images of cytotoxic T cells In the left column cell surface antigens were stained for CD3(green FITC) and the staining of CD8 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI (blue)and the last column presents a colocalized fluorescence image of CD3+CD8+ T cells

Acknowledgments

The authors would like to thank Man-Hui Pai Assistant Pro-fessor of Department of Anatomy Taipei Medical Universityfor her technical assistance in immunofluorescence stainingThis studywas supported by aResearchGrant (NSC100-2320-B-038-007-MY3) from the National Science Council TaipeiTaiwan

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] C T Murphy K Nally F Shanahan and S Melgar ldquoShining alight on intestinal trafficrdquoClinical and Developmental Immunol-ogy vol 2012 Article ID 808157 14 pages 2012

Mediators of Inflammation 13

[3] L A Zenewicz A Antov and R A Flavell ldquoCD4 T-cell differ-entiation and inflammatory bowel diseaserdquo Trends inMolecularMedicine vol 15 no 5 pp 199ndash207 2009

[4] J C Lee P A Lyons E F McKinney et al ldquoGene expressionprofiling of CD8+ T cells predicts prognosis in patients withCrohn disease and ulcerative colitisrdquo Journal of Clinical Inves-tigation vol 121 no 10 pp 4170ndash4179 2011

[5] N T Funderburg S R Stubblefield Park H C Sung et alldquoCirculating CD4+ and CD8+ T cells are activated in IBDand are associated with plasma markers of inflammationrdquoImmunology vol 140 no 1 pp 87ndash97 2013

[6] E J Villablanca B Cassani U H von Andrian and J RMora ldquoBlocking lymphocyte localization to the gastrointestinalmucosa as a therapeutic strategy for inflammatory boweldiseasesrdquo Gastroenterology vol 140 no 6 pp 1776ndash1784 2011

[7] R P McEver and R D Cummings ldquoPerspectives series celladhesion in vascular biology Role of PSGL-1 binding toselectins in leukocyte recruitmentrdquo The Journal of ClinicalInvestigation vol 100 no 3 pp 485ndash491 1997

[8] C Berlin E L Berg M J Briskin et al ldquo12057241205737 Integrin medi-ates lymphocyte binding to the mucosal vascular addressinMAdCAM-1rdquo Cell vol 74 no 1 pp 185ndash195 1993

[9] K Ley C Laudanna M I Cybulsky and S Nourshargh ldquoGet-ting to the site of inflammation the leukocyte adhesion cascadeupdatedrdquoNature Reviews Immunology vol 7 no 9 pp 678ndash6892007

[10] B Moser M Wolf A Walz and P Loetscher ldquoChemokinesmultiple levels of leukocyte migration controlrdquo Trends inImmunology vol 25 no 2 pp 75ndash84 2004

[11] S Arimilli W Ferlin N Solvason S Deshpande M Howardand S Mocci ldquoChemokines in autoimmune diseasesrdquo Immuno-logical Reviews vol 177 pp 43ndash51 2000

[12] C Koenecke and R Forster ldquoCCR9 and inflammatory boweldiseaserdquo Expert Opinion on Therapeutic Targets vol 13 no 3pp 297ndash306 2009

[13] B Johansson-Lindbom and W W Agace ldquoGeneration of gut-homing T cells and their localization to the small intestinalmucosardquo Immunological Reviews vol 215 no 1 pp 226ndash2422007

[14] P EWischmeyer ldquoGlutaminemode of action in critical illnessrdquoCritical Care Medicine vol 35 no 9 pp S541ndashS544 2007

[15] N A Kretzmann H Fillmann J L Mauriz C A MarroniJ Gonzalez-Gallego and M J Tunon ldquoEffects of glutamineon proinflammatory gene expression and activation of nuclearfactor Kappa B and signal transducers and activators oftranscription in TNBS-induced colitisrdquo Inflammatory BowelDiseases vol 14 no 11 pp 1504ndash1513 2008

[16] H Xue A J D Sufit and P EWischmeyer ldquoGlutamine therapyimproves outcome of in vitro and in vivo experimental colitismodelsrdquo Journal of Parenteral and Enteral Nutrition vol 35 no2 pp 188ndash197 2011

[17] C-C Chu Y-C Hou M-H Pai C-J Chao and S-L YehldquoPretreatment with alanyl-glutamine suppresses T-helper-cell-associated cytokine expression and reduces inflammatoryresponses in mice with acute DSS-induced colitisrdquo The Journalof Nutritional Biochemistry vol 23 no 9 pp 1092ndash1099 2011

[18] S Kew S M Wells P Yaqoob F A Wallace E A Miles and PC Calder ldquoDietary glutamine enhances murine T-lymphocyteresponsivenessrdquo The Journal of Nutrition vol 129 no 8 pp1524ndash1531 1999

[19] S M Wells S Kew P Yaqoob F A Wallace and P C CalderldquoDietary glutamine enhances cytokine production by murinemacrophagesrdquo Nutrition vol 15 no 11-12 pp 881ndash884 1999

[20] Y-C Hou M-H Pai W-C Chiu Y-M Hu and S-L YehldquoEffects of dietary glutamine supplementation on lung injuryinduced by lipopolysaccharide administrationrdquo American Jour-nal of PhysiologymdashLung Cellular and Molecular Physiology vol296 no 3 pp L288ndashL295 2009

[21] M Kawada A Arihiro and E Mizoguchi ldquoInsights fromadvances in research of chemically induced experimental mod-els of human inflammatory bowel diseaserdquo World Journal ofGastroenterology vol 13 no 42 pp 5581ndash5593 2007

[22] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[23] R Yazbeck G S Howarth R N Butler M S Geier and CA Abbott ldquoBiochemical and histological changes in the smallintestine of mice with dextran sulfate sodium colitisrdquo Journal ofCellular Physiology vol 226 no 12 pp 3219ndash3224 2011

[24] M Mahler I J Bristol E H Leiter et al ldquoDifferentialsusceptibility of inbred mouse strains to dextran sulfatesodium-induced colitisrdquo American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 274 no 3 part 1 ppG544ndashG551 1998

[25] S Melgar A Karlsson and E Michaelsson ldquoAcute colitisinduced by dextran sulfate sodium progresses to chronicityin C57BL6 but not in BALBc mice correlation betweensymptoms and inflammationrdquoAmerican Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 288 no 6 pp G1328ndashG1338 2005

[26] MKoizumiN King R Lobb C Benjamin andDK PodolskyldquoExpression of vascular adhesion molecules in inflammatorybowel diseaserdquo Gastroenterology vol 103 no 3 pp 840ndash8471992

[27] L Peyrin-Biroulet E V Loftus Jr J-F Colombel and W JSandborn ldquoLong-term complications extraintestinal manifes-tations and mortality in adult Crohnrsquos disease in population-based cohortsrdquo Inflammatory Bowel Diseases vol 17 no 1 pp471ndash478 2011

[28] D H Adams and B Eksteen ldquoAberrant homing of mucosal Tcells and extra-intestinal manifestations of inflammatory boweldiseaserdquoNature Reviews Immunology vol 6 no 3 pp 244ndash2512006

[29] TW Spahn H Herbst P D Rennert et al ldquoInduction of colitisinmice deficient of Peyerrsquos patches andmesenteric lymph nodesis associated with increased disease severity and formation ofcolonic lymphoid patchesrdquo The American Journal of Pathologyvol 161 no 6 pp 2273ndash2282 2002

[30] S Nancey S Holvoet I Graber et al ldquoCD8+ Cytotoxic T CellsInduce Relapsing Colitis in Normal Micerdquo Gastroenterologyvol 131 no 2 pp 485ndash496 2006

[31] E C Butcher and L J Picker ldquoLymphocyte homing and home-ostasisrdquo Science vol 272 no 5258 pp 60ndash66 1996

[32] C Berlin R F Bargatze J J Campbell et al ldquo1205724 Integrinsmediate lymphocyte attachment and rolling under physiologicflowrdquo Cell vol 80 no 3 pp 413ndash422 1995

[33] K A Papadakis J Prehn V Nelson et al ldquoThe role of thymus-expressed chemokine and its receptor CCR9 on lymphocytesin the regional specialization of the mucosal immune systemrdquoJournal of Immunology vol 165 no 9 pp 5069ndash5076 2000

14 Mediators of Inflammation

[34] M-A Wurbel M G McIntire P Dwyer and E FiebigerldquoCCL25CCR9 interactions regulate large intestinal inflamma-tion in a murine model of acute colitisrdquo PLoS ONE vol 6 no 1Article ID e16442 2011

[35] CWang E K Hanly LWWheeler M Kaur K GMcDonaldand R D Newberry ldquoEffect of 12057241205737 blockade on intestinallymphocyte subsets and lymphoid tissue developmentrdquo Inflam-matory Bowel Diseases vol 16 no 10 pp 1751ndash1762 2010

[36] M Coeffier R Marion-Letellier and P Dechelotte ldquoPotentialfor amino acids supplementation during inflammatory boweldiseasesrdquo Inflammatory Bowel Diseases vol 16 no 3 pp 518ndash524 2010

[37] B San-Miguel I Crespo N A Kretzmann et al ldquoGlutamineprevents fibrosis development in rats with colitis induced by246-trinitrobenzene sulfonic acidrdquo The Journal of Nutritionvol 140 no 6 pp 1065ndash1071 2010

[38] Y C Hou C C Chu T L Ko et al ldquoEffects of alanyl-glutaminedipeptide on the expression of colon-inflammatory mediatorsduring the recovery phase of colitis induced by dextran sulfatesodiumrdquo European Journal of Nutrition vol 52 no 3 pp 1089ndash1098 2013

[39] Y Shiomi S Nishiumi M Ooi et al ldquoGCMS-based meta-bolomic study in mice with colitis induced by dextran sulfatesodiumrdquo Inflammatory Bowel Diseases vol 17 no 11 pp 2261ndash2274 2011

[40] M Vicario C Amat M Rivero M Moreto and C PelegrıldquoDietary glutamine affects mucosal functions in rats with mildDSS-induced colitisrdquoThe Journal of Nutrition vol 137 no 8 pp1931ndash1937 2007

[41] H Arndt F Kullmann F Reuszlig J Scholmerich and K-D Pal-itzsch ldquoGlutamine attenuates leukocyte-endothelial cell adhe-sion in indomethacin-induced intestinal inflammation in theratrdquo Journal of Parenteral and Enteral Nutrition vol 23 no 1pp 12ndash18 1999

[42] P Yaqoob and P C Calder ldquoGlutamine requirement of prolif-erating T lymphocytesrdquo Nutrition vol 13 no 7-8 pp 646ndash6511997

[43] E L Carr A Kelman G S Wu et al ldquoGlutamine uptake andmetabolism are coordinately regulated by ERKMAPK duringT lymphocyte activationrdquo Journal of Immunology vol 185 no2 pp 1037ndash1044 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

12 Mediators of Inflammation

50120583m

CD3

50120583m

50120583m

50120583m

C

G

DC

DG

(a)

CD8

50120583m

50120583m

50120583m

50120583m

(b)

50120583m

50120583m

50120583m

50120583m

Merge

(c)

Figure 10 Representative immunofluorescence images of cytotoxic T cells In the left column cell surface antigens were stained for CD3(green FITC) and the staining of CD8 cells (red rhodamine) is illustrated in the middle column The nucleus is stained with DAPI (blue)and the last column presents a colocalized fluorescence image of CD3+CD8+ T cells

Acknowledgments

The authors would like to thank Man-Hui Pai Assistant Pro-fessor of Department of Anatomy Taipei Medical Universityfor her technical assistance in immunofluorescence stainingThis studywas supported by aResearchGrant (NSC100-2320-B-038-007-MY3) from the National Science Council TaipeiTaiwan

References

[1] A Kaser S Zeissig and R S Blumberg ldquoInflammatory boweldiseaserdquo Annual Review of Immunology vol 28 pp 573ndash6212010

[2] C T Murphy K Nally F Shanahan and S Melgar ldquoShining alight on intestinal trafficrdquoClinical and Developmental Immunol-ogy vol 2012 Article ID 808157 14 pages 2012

Mediators of Inflammation 13

[3] L A Zenewicz A Antov and R A Flavell ldquoCD4 T-cell differ-entiation and inflammatory bowel diseaserdquo Trends inMolecularMedicine vol 15 no 5 pp 199ndash207 2009

[4] J C Lee P A Lyons E F McKinney et al ldquoGene expressionprofiling of CD8+ T cells predicts prognosis in patients withCrohn disease and ulcerative colitisrdquo Journal of Clinical Inves-tigation vol 121 no 10 pp 4170ndash4179 2011

[5] N T Funderburg S R Stubblefield Park H C Sung et alldquoCirculating CD4+ and CD8+ T cells are activated in IBDand are associated with plasma markers of inflammationrdquoImmunology vol 140 no 1 pp 87ndash97 2013

[6] E J Villablanca B Cassani U H von Andrian and J RMora ldquoBlocking lymphocyte localization to the gastrointestinalmucosa as a therapeutic strategy for inflammatory boweldiseasesrdquo Gastroenterology vol 140 no 6 pp 1776ndash1784 2011

[7] R P McEver and R D Cummings ldquoPerspectives series celladhesion in vascular biology Role of PSGL-1 binding toselectins in leukocyte recruitmentrdquo The Journal of ClinicalInvestigation vol 100 no 3 pp 485ndash491 1997

[8] C Berlin E L Berg M J Briskin et al ldquo12057241205737 Integrin medi-ates lymphocyte binding to the mucosal vascular addressinMAdCAM-1rdquo Cell vol 74 no 1 pp 185ndash195 1993

[9] K Ley C Laudanna M I Cybulsky and S Nourshargh ldquoGet-ting to the site of inflammation the leukocyte adhesion cascadeupdatedrdquoNature Reviews Immunology vol 7 no 9 pp 678ndash6892007

[10] B Moser M Wolf A Walz and P Loetscher ldquoChemokinesmultiple levels of leukocyte migration controlrdquo Trends inImmunology vol 25 no 2 pp 75ndash84 2004

[11] S Arimilli W Ferlin N Solvason S Deshpande M Howardand S Mocci ldquoChemokines in autoimmune diseasesrdquo Immuno-logical Reviews vol 177 pp 43ndash51 2000

[12] C Koenecke and R Forster ldquoCCR9 and inflammatory boweldiseaserdquo Expert Opinion on Therapeutic Targets vol 13 no 3pp 297ndash306 2009

[13] B Johansson-Lindbom and W W Agace ldquoGeneration of gut-homing T cells and their localization to the small intestinalmucosardquo Immunological Reviews vol 215 no 1 pp 226ndash2422007

[14] P EWischmeyer ldquoGlutaminemode of action in critical illnessrdquoCritical Care Medicine vol 35 no 9 pp S541ndashS544 2007

[15] N A Kretzmann H Fillmann J L Mauriz C A MarroniJ Gonzalez-Gallego and M J Tunon ldquoEffects of glutamineon proinflammatory gene expression and activation of nuclearfactor Kappa B and signal transducers and activators oftranscription in TNBS-induced colitisrdquo Inflammatory BowelDiseases vol 14 no 11 pp 1504ndash1513 2008

[16] H Xue A J D Sufit and P EWischmeyer ldquoGlutamine therapyimproves outcome of in vitro and in vivo experimental colitismodelsrdquo Journal of Parenteral and Enteral Nutrition vol 35 no2 pp 188ndash197 2011

[17] C-C Chu Y-C Hou M-H Pai C-J Chao and S-L YehldquoPretreatment with alanyl-glutamine suppresses T-helper-cell-associated cytokine expression and reduces inflammatoryresponses in mice with acute DSS-induced colitisrdquo The Journalof Nutritional Biochemistry vol 23 no 9 pp 1092ndash1099 2011

[18] S Kew S M Wells P Yaqoob F A Wallace E A Miles and PC Calder ldquoDietary glutamine enhances murine T-lymphocyteresponsivenessrdquo The Journal of Nutrition vol 129 no 8 pp1524ndash1531 1999

[19] S M Wells S Kew P Yaqoob F A Wallace and P C CalderldquoDietary glutamine enhances cytokine production by murinemacrophagesrdquo Nutrition vol 15 no 11-12 pp 881ndash884 1999

[20] Y-C Hou M-H Pai W-C Chiu Y-M Hu and S-L YehldquoEffects of dietary glutamine supplementation on lung injuryinduced by lipopolysaccharide administrationrdquo American Jour-nal of PhysiologymdashLung Cellular and Molecular Physiology vol296 no 3 pp L288ndashL295 2009

[21] M Kawada A Arihiro and E Mizoguchi ldquoInsights fromadvances in research of chemically induced experimental mod-els of human inflammatory bowel diseaserdquo World Journal ofGastroenterology vol 13 no 42 pp 5581ndash5593 2007

[22] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[23] R Yazbeck G S Howarth R N Butler M S Geier and CA Abbott ldquoBiochemical and histological changes in the smallintestine of mice with dextran sulfate sodium colitisrdquo Journal ofCellular Physiology vol 226 no 12 pp 3219ndash3224 2011

[24] M Mahler I J Bristol E H Leiter et al ldquoDifferentialsusceptibility of inbred mouse strains to dextran sulfatesodium-induced colitisrdquo American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 274 no 3 part 1 ppG544ndashG551 1998

[25] S Melgar A Karlsson and E Michaelsson ldquoAcute colitisinduced by dextran sulfate sodium progresses to chronicityin C57BL6 but not in BALBc mice correlation betweensymptoms and inflammationrdquoAmerican Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 288 no 6 pp G1328ndashG1338 2005

[26] MKoizumiN King R Lobb C Benjamin andDK PodolskyldquoExpression of vascular adhesion molecules in inflammatorybowel diseaserdquo Gastroenterology vol 103 no 3 pp 840ndash8471992

[27] L Peyrin-Biroulet E V Loftus Jr J-F Colombel and W JSandborn ldquoLong-term complications extraintestinal manifes-tations and mortality in adult Crohnrsquos disease in population-based cohortsrdquo Inflammatory Bowel Diseases vol 17 no 1 pp471ndash478 2011

[28] D H Adams and B Eksteen ldquoAberrant homing of mucosal Tcells and extra-intestinal manifestations of inflammatory boweldiseaserdquoNature Reviews Immunology vol 6 no 3 pp 244ndash2512006

[29] TW Spahn H Herbst P D Rennert et al ldquoInduction of colitisinmice deficient of Peyerrsquos patches andmesenteric lymph nodesis associated with increased disease severity and formation ofcolonic lymphoid patchesrdquo The American Journal of Pathologyvol 161 no 6 pp 2273ndash2282 2002

[30] S Nancey S Holvoet I Graber et al ldquoCD8+ Cytotoxic T CellsInduce Relapsing Colitis in Normal Micerdquo Gastroenterologyvol 131 no 2 pp 485ndash496 2006

[31] E C Butcher and L J Picker ldquoLymphocyte homing and home-ostasisrdquo Science vol 272 no 5258 pp 60ndash66 1996

[32] C Berlin R F Bargatze J J Campbell et al ldquo1205724 Integrinsmediate lymphocyte attachment and rolling under physiologicflowrdquo Cell vol 80 no 3 pp 413ndash422 1995

[33] K A Papadakis J Prehn V Nelson et al ldquoThe role of thymus-expressed chemokine and its receptor CCR9 on lymphocytesin the regional specialization of the mucosal immune systemrdquoJournal of Immunology vol 165 no 9 pp 5069ndash5076 2000

14 Mediators of Inflammation

[34] M-A Wurbel M G McIntire P Dwyer and E FiebigerldquoCCL25CCR9 interactions regulate large intestinal inflamma-tion in a murine model of acute colitisrdquo PLoS ONE vol 6 no 1Article ID e16442 2011

[35] CWang E K Hanly LWWheeler M Kaur K GMcDonaldand R D Newberry ldquoEffect of 12057241205737 blockade on intestinallymphocyte subsets and lymphoid tissue developmentrdquo Inflam-matory Bowel Diseases vol 16 no 10 pp 1751ndash1762 2010

[36] M Coeffier R Marion-Letellier and P Dechelotte ldquoPotentialfor amino acids supplementation during inflammatory boweldiseasesrdquo Inflammatory Bowel Diseases vol 16 no 3 pp 518ndash524 2010

[37] B San-Miguel I Crespo N A Kretzmann et al ldquoGlutamineprevents fibrosis development in rats with colitis induced by246-trinitrobenzene sulfonic acidrdquo The Journal of Nutritionvol 140 no 6 pp 1065ndash1071 2010

[38] Y C Hou C C Chu T L Ko et al ldquoEffects of alanyl-glutaminedipeptide on the expression of colon-inflammatory mediatorsduring the recovery phase of colitis induced by dextran sulfatesodiumrdquo European Journal of Nutrition vol 52 no 3 pp 1089ndash1098 2013

[39] Y Shiomi S Nishiumi M Ooi et al ldquoGCMS-based meta-bolomic study in mice with colitis induced by dextran sulfatesodiumrdquo Inflammatory Bowel Diseases vol 17 no 11 pp 2261ndash2274 2011

[40] M Vicario C Amat M Rivero M Moreto and C PelegrıldquoDietary glutamine affects mucosal functions in rats with mildDSS-induced colitisrdquoThe Journal of Nutrition vol 137 no 8 pp1931ndash1937 2007

[41] H Arndt F Kullmann F Reuszlig J Scholmerich and K-D Pal-itzsch ldquoGlutamine attenuates leukocyte-endothelial cell adhe-sion in indomethacin-induced intestinal inflammation in theratrdquo Journal of Parenteral and Enteral Nutrition vol 23 no 1pp 12ndash18 1999

[42] P Yaqoob and P C Calder ldquoGlutamine requirement of prolif-erating T lymphocytesrdquo Nutrition vol 13 no 7-8 pp 646ndash6511997

[43] E L Carr A Kelman G S Wu et al ldquoGlutamine uptake andmetabolism are coordinately regulated by ERKMAPK duringT lymphocyte activationrdquo Journal of Immunology vol 185 no2 pp 1037ndash1044 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 13

[3] L A Zenewicz A Antov and R A Flavell ldquoCD4 T-cell differ-entiation and inflammatory bowel diseaserdquo Trends inMolecularMedicine vol 15 no 5 pp 199ndash207 2009

[4] J C Lee P A Lyons E F McKinney et al ldquoGene expressionprofiling of CD8+ T cells predicts prognosis in patients withCrohn disease and ulcerative colitisrdquo Journal of Clinical Inves-tigation vol 121 no 10 pp 4170ndash4179 2011

[5] N T Funderburg S R Stubblefield Park H C Sung et alldquoCirculating CD4+ and CD8+ T cells are activated in IBDand are associated with plasma markers of inflammationrdquoImmunology vol 140 no 1 pp 87ndash97 2013

[6] E J Villablanca B Cassani U H von Andrian and J RMora ldquoBlocking lymphocyte localization to the gastrointestinalmucosa as a therapeutic strategy for inflammatory boweldiseasesrdquo Gastroenterology vol 140 no 6 pp 1776ndash1784 2011

[7] R P McEver and R D Cummings ldquoPerspectives series celladhesion in vascular biology Role of PSGL-1 binding toselectins in leukocyte recruitmentrdquo The Journal of ClinicalInvestigation vol 100 no 3 pp 485ndash491 1997

[8] C Berlin E L Berg M J Briskin et al ldquo12057241205737 Integrin medi-ates lymphocyte binding to the mucosal vascular addressinMAdCAM-1rdquo Cell vol 74 no 1 pp 185ndash195 1993

[9] K Ley C Laudanna M I Cybulsky and S Nourshargh ldquoGet-ting to the site of inflammation the leukocyte adhesion cascadeupdatedrdquoNature Reviews Immunology vol 7 no 9 pp 678ndash6892007

[10] B Moser M Wolf A Walz and P Loetscher ldquoChemokinesmultiple levels of leukocyte migration controlrdquo Trends inImmunology vol 25 no 2 pp 75ndash84 2004

[11] S Arimilli W Ferlin N Solvason S Deshpande M Howardand S Mocci ldquoChemokines in autoimmune diseasesrdquo Immuno-logical Reviews vol 177 pp 43ndash51 2000

[12] C Koenecke and R Forster ldquoCCR9 and inflammatory boweldiseaserdquo Expert Opinion on Therapeutic Targets vol 13 no 3pp 297ndash306 2009

[13] B Johansson-Lindbom and W W Agace ldquoGeneration of gut-homing T cells and their localization to the small intestinalmucosardquo Immunological Reviews vol 215 no 1 pp 226ndash2422007

[14] P EWischmeyer ldquoGlutaminemode of action in critical illnessrdquoCritical Care Medicine vol 35 no 9 pp S541ndashS544 2007

[15] N A Kretzmann H Fillmann J L Mauriz C A MarroniJ Gonzalez-Gallego and M J Tunon ldquoEffects of glutamineon proinflammatory gene expression and activation of nuclearfactor Kappa B and signal transducers and activators oftranscription in TNBS-induced colitisrdquo Inflammatory BowelDiseases vol 14 no 11 pp 1504ndash1513 2008

[16] H Xue A J D Sufit and P EWischmeyer ldquoGlutamine therapyimproves outcome of in vitro and in vivo experimental colitismodelsrdquo Journal of Parenteral and Enteral Nutrition vol 35 no2 pp 188ndash197 2011

[17] C-C Chu Y-C Hou M-H Pai C-J Chao and S-L YehldquoPretreatment with alanyl-glutamine suppresses T-helper-cell-associated cytokine expression and reduces inflammatoryresponses in mice with acute DSS-induced colitisrdquo The Journalof Nutritional Biochemistry vol 23 no 9 pp 1092ndash1099 2011

[18] S Kew S M Wells P Yaqoob F A Wallace E A Miles and PC Calder ldquoDietary glutamine enhances murine T-lymphocyteresponsivenessrdquo The Journal of Nutrition vol 129 no 8 pp1524ndash1531 1999

[19] S M Wells S Kew P Yaqoob F A Wallace and P C CalderldquoDietary glutamine enhances cytokine production by murinemacrophagesrdquo Nutrition vol 15 no 11-12 pp 881ndash884 1999

[20] Y-C Hou M-H Pai W-C Chiu Y-M Hu and S-L YehldquoEffects of dietary glutamine supplementation on lung injuryinduced by lipopolysaccharide administrationrdquo American Jour-nal of PhysiologymdashLung Cellular and Molecular Physiology vol296 no 3 pp L288ndashL295 2009

[21] M Kawada A Arihiro and E Mizoguchi ldquoInsights fromadvances in research of chemically induced experimental mod-els of human inflammatory bowel diseaserdquo World Journal ofGastroenterology vol 13 no 42 pp 5581ndash5593 2007

[22] I Okayasu S Hatakeyama M Yamada T Ohkusa Y Inagakiand R Nakaya ldquoA novel method in the induction of reliableexperimental acute and chronic ulcerative colitis in micerdquoGastroenterology vol 98 no 3 pp 694ndash702 1990

[23] R Yazbeck G S Howarth R N Butler M S Geier and CA Abbott ldquoBiochemical and histological changes in the smallintestine of mice with dextran sulfate sodium colitisrdquo Journal ofCellular Physiology vol 226 no 12 pp 3219ndash3224 2011

[24] M Mahler I J Bristol E H Leiter et al ldquoDifferentialsusceptibility of inbred mouse strains to dextran sulfatesodium-induced colitisrdquo American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 274 no 3 part 1 ppG544ndashG551 1998

[25] S Melgar A Karlsson and E Michaelsson ldquoAcute colitisinduced by dextran sulfate sodium progresses to chronicityin C57BL6 but not in BALBc mice correlation betweensymptoms and inflammationrdquoAmerican Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 288 no 6 pp G1328ndashG1338 2005

[26] MKoizumiN King R Lobb C Benjamin andDK PodolskyldquoExpression of vascular adhesion molecules in inflammatorybowel diseaserdquo Gastroenterology vol 103 no 3 pp 840ndash8471992

[27] L Peyrin-Biroulet E V Loftus Jr J-F Colombel and W JSandborn ldquoLong-term complications extraintestinal manifes-tations and mortality in adult Crohnrsquos disease in population-based cohortsrdquo Inflammatory Bowel Diseases vol 17 no 1 pp471ndash478 2011

[28] D H Adams and B Eksteen ldquoAberrant homing of mucosal Tcells and extra-intestinal manifestations of inflammatory boweldiseaserdquoNature Reviews Immunology vol 6 no 3 pp 244ndash2512006

[29] TW Spahn H Herbst P D Rennert et al ldquoInduction of colitisinmice deficient of Peyerrsquos patches andmesenteric lymph nodesis associated with increased disease severity and formation ofcolonic lymphoid patchesrdquo The American Journal of Pathologyvol 161 no 6 pp 2273ndash2282 2002

[30] S Nancey S Holvoet I Graber et al ldquoCD8+ Cytotoxic T CellsInduce Relapsing Colitis in Normal Micerdquo Gastroenterologyvol 131 no 2 pp 485ndash496 2006

[31] E C Butcher and L J Picker ldquoLymphocyte homing and home-ostasisrdquo Science vol 272 no 5258 pp 60ndash66 1996

[32] C Berlin R F Bargatze J J Campbell et al ldquo1205724 Integrinsmediate lymphocyte attachment and rolling under physiologicflowrdquo Cell vol 80 no 3 pp 413ndash422 1995

[33] K A Papadakis J Prehn V Nelson et al ldquoThe role of thymus-expressed chemokine and its receptor CCR9 on lymphocytesin the regional specialization of the mucosal immune systemrdquoJournal of Immunology vol 165 no 9 pp 5069ndash5076 2000

14 Mediators of Inflammation

[34] M-A Wurbel M G McIntire P Dwyer and E FiebigerldquoCCL25CCR9 interactions regulate large intestinal inflamma-tion in a murine model of acute colitisrdquo PLoS ONE vol 6 no 1Article ID e16442 2011

[35] CWang E K Hanly LWWheeler M Kaur K GMcDonaldand R D Newberry ldquoEffect of 12057241205737 blockade on intestinallymphocyte subsets and lymphoid tissue developmentrdquo Inflam-matory Bowel Diseases vol 16 no 10 pp 1751ndash1762 2010

[36] M Coeffier R Marion-Letellier and P Dechelotte ldquoPotentialfor amino acids supplementation during inflammatory boweldiseasesrdquo Inflammatory Bowel Diseases vol 16 no 3 pp 518ndash524 2010

[37] B San-Miguel I Crespo N A Kretzmann et al ldquoGlutamineprevents fibrosis development in rats with colitis induced by246-trinitrobenzene sulfonic acidrdquo The Journal of Nutritionvol 140 no 6 pp 1065ndash1071 2010

[38] Y C Hou C C Chu T L Ko et al ldquoEffects of alanyl-glutaminedipeptide on the expression of colon-inflammatory mediatorsduring the recovery phase of colitis induced by dextran sulfatesodiumrdquo European Journal of Nutrition vol 52 no 3 pp 1089ndash1098 2013

[39] Y Shiomi S Nishiumi M Ooi et al ldquoGCMS-based meta-bolomic study in mice with colitis induced by dextran sulfatesodiumrdquo Inflammatory Bowel Diseases vol 17 no 11 pp 2261ndash2274 2011

[40] M Vicario C Amat M Rivero M Moreto and C PelegrıldquoDietary glutamine affects mucosal functions in rats with mildDSS-induced colitisrdquoThe Journal of Nutrition vol 137 no 8 pp1931ndash1937 2007

[41] H Arndt F Kullmann F Reuszlig J Scholmerich and K-D Pal-itzsch ldquoGlutamine attenuates leukocyte-endothelial cell adhe-sion in indomethacin-induced intestinal inflammation in theratrdquo Journal of Parenteral and Enteral Nutrition vol 23 no 1pp 12ndash18 1999

[42] P Yaqoob and P C Calder ldquoGlutamine requirement of prolif-erating T lymphocytesrdquo Nutrition vol 13 no 7-8 pp 646ndash6511997

[43] E L Carr A Kelman G S Wu et al ldquoGlutamine uptake andmetabolism are coordinately regulated by ERKMAPK duringT lymphocyte activationrdquo Journal of Immunology vol 185 no2 pp 1037ndash1044 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

14 Mediators of Inflammation

[34] M-A Wurbel M G McIntire P Dwyer and E FiebigerldquoCCL25CCR9 interactions regulate large intestinal inflamma-tion in a murine model of acute colitisrdquo PLoS ONE vol 6 no 1Article ID e16442 2011

[35] CWang E K Hanly LWWheeler M Kaur K GMcDonaldand R D Newberry ldquoEffect of 12057241205737 blockade on intestinallymphocyte subsets and lymphoid tissue developmentrdquo Inflam-matory Bowel Diseases vol 16 no 10 pp 1751ndash1762 2010

[36] M Coeffier R Marion-Letellier and P Dechelotte ldquoPotentialfor amino acids supplementation during inflammatory boweldiseasesrdquo Inflammatory Bowel Diseases vol 16 no 3 pp 518ndash524 2010

[37] B San-Miguel I Crespo N A Kretzmann et al ldquoGlutamineprevents fibrosis development in rats with colitis induced by246-trinitrobenzene sulfonic acidrdquo The Journal of Nutritionvol 140 no 6 pp 1065ndash1071 2010

[38] Y C Hou C C Chu T L Ko et al ldquoEffects of alanyl-glutaminedipeptide on the expression of colon-inflammatory mediatorsduring the recovery phase of colitis induced by dextran sulfatesodiumrdquo European Journal of Nutrition vol 52 no 3 pp 1089ndash1098 2013

[39] Y Shiomi S Nishiumi M Ooi et al ldquoGCMS-based meta-bolomic study in mice with colitis induced by dextran sulfatesodiumrdquo Inflammatory Bowel Diseases vol 17 no 11 pp 2261ndash2274 2011

[40] M Vicario C Amat M Rivero M Moreto and C PelegrıldquoDietary glutamine affects mucosal functions in rats with mildDSS-induced colitisrdquoThe Journal of Nutrition vol 137 no 8 pp1931ndash1937 2007

[41] H Arndt F Kullmann F Reuszlig J Scholmerich and K-D Pal-itzsch ldquoGlutamine attenuates leukocyte-endothelial cell adhe-sion in indomethacin-induced intestinal inflammation in theratrdquo Journal of Parenteral and Enteral Nutrition vol 23 no 1pp 12ndash18 1999

[42] P Yaqoob and P C Calder ldquoGlutamine requirement of prolif-erating T lymphocytesrdquo Nutrition vol 13 no 7-8 pp 646ndash6511997

[43] E L Carr A Kelman G S Wu et al ldquoGlutamine uptake andmetabolism are coordinately regulated by ERKMAPK duringT lymphocyte activationrdquo Journal of Immunology vol 185 no2 pp 1037ndash1044 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom